MCB4403 Level 2-3 Studying Final Exam

What are phylogenetics in connection to microorganisms, and how are microorganisms sorted phylogenetically?

Phylogenetics is the classification system for organisms based on their evolutionary relationship. Microorganisms are sorted phylogenetically primarily through the analysis of nucleic acid sequences, most notably the sequencing of the 16S ribosomal RNA genes. This molecular approach allows scientists to classify all life into three overarching domains: Archaea, Bacteria, and Eukarya.

What is the endosymbiotic theory?

The Endosymbiotic Theory is the idea that specialized membrane-bound organelles found in eukaryotic cells, such as mitochondria and chloroplasts, originated when ancestral cells engulfed small prokaryotes. Over evolutionary time, these internalized prokaryotes lost their ability to exist independently, becoming the essential organelles we recognize today.

What are endotoxins and what purpose do they serve?

Endotoxins are toxic molecules found as part of the bacterial cell itself. They are lipopolysaccharides (LPS) that make up the outer layer of the Gram-negative bacterial outer membrane. The Lipid A component of the LPS contributes most of the toxic effects. Endotoxins are mainly released when the Gram-negative cell dies and the cell wall fragments. Once released into the bloodstream, they can induce macrophages to produce inflammatory molecules, potentially leading to fever, inflammation, hemorrhaging, and septic shock.

What is the periplasmic space, what microorganisms have it, and what purpose does it serve?

The periplasmic space is the region located between the plasma membrane and the outer membrane/cell wall in certain bacteria. This space is primarily found in Gram-negative bacteria. Its purpose is to act as a site of metabolic activity, containing various enzymes and transport proteins necessary for cellular function.

What are the structural and functional differences between flagella, pili, and cilia?

Bacterial flagella are long, helical protein filaments used for motility. They function like propellers, rotating to push the cell; rotation is powered by the proton motive force (PMF). Bacterial pili (also called fimbriae) are short, hair-like appendages used for attachment to surfaces, often carrying adhesins for specific host receptors. Pili are not involved in motility, though a specific type, the sex pilus, is essential for conjugation (genetic transfer). Cilia and eukaryotic flagella are structurally complex, built from a "9+2" arrangement of microtubules. Eukaryotic flagella propel the cell with a wave-like beating motion (not rotation), while cilia are shorter and typically beat in coordinated fashion, often found in large numbers on a cell's surface.

What are the major differences physiologically between prokaryotic and eukaryotic cells?

Prokaryotic and eukaryotic cells differ fundamentally in their physiology and structure. Prokaryotes are typically much smaller and lack a nucleus and membrane-bound organelles. They benefit from miniaturization, allowing for high metabolic activity and fast replication rates. Eukaryotes are larger, possess a true membrane-bound nucleus and organized internal compartments (like mitochondria and ER). This compartmentalization isolates metabolic activities, allowing reactions to proceed faster and maintaining different conditions (like acidity in lysosomes). For energy production, prokaryotes use respiratory enzymes associated with their plasma membrane, whereas eukaryotes localize this activity primarily within their mitochondria. Furthermore, eukaryotes can ingest large particles via endocytosis, a process generally absent in prokaryotes.

How does prokaryotic and eukaryotic cell DNA differ, and how does their replication differ as well?

Prokaryotic and eukaryotic DNA differ significantly: prokaryotes usually have a single, circular chromosome (nucleoid) that lacks histones. Eukaryotes possess multiple, linear chromosomes that are highly organized and condensed around histone proteins. Their replication mechanisms also differ: prokaryotic replication begins at a single origin and proceeds bidirectionally, forming one replication bubble. Eukaryotic replication initiates at numerous origins simultaneously along each linear chromosome.

What are plasmids and what purpose do they serve?

Plasmids are small, extra-chromosomal pieces of DNA typically found as closed circular molecules in bacteria. They replicate independently of the main bacterial chromosome and are inherited by daughter cells. Plasmids are non-essential for survival but carry genes that provide a selective advantage to the host, such as antibiotic resistance or the ability to produce toxins.

What is the structure of a ribosome, and how does it differ between prokaryotes and eukaryotes?

The ribosome is the cellular machinery composed of protein and RNA that acts as the site of protein synthesis. Ribosomes are measured in Svedberg units (S). The prokaryotic ribosome is smaller (70S), comprised of a 50S large subunit and a 30S small subunit. In contrast, the eukaryotic ribosome is larger (80S), built from a 60S large subunit and a 40S small subunit.

What is the structure of the plasma membrane? What components are imbedded in the membrane and what functions do they serve?

The plasma membrane is a phospholipid bilayer that surrounds the cytoplasm. It acts as a selectively permeable barrier that regulates the movement of substances in and out of the cell. Phospholipids form the bulk of the barrier, arranging with their hydrophilic heads facing outward and hydrophobic tails forming the interior. Embedded within this lipid structure are various proteins (integral and peripheral). These proteins are critical for function, facilitating active transport of nutrients and carrying enzymes involved in crucial metabolic processes.

What are the structural differences between gram-positive and gram-negative cell walls? What is the order, from inside to outside, of each cell wall component for both kinds?

The two main types of bacterial cell walls, Gram-positive and Gram-negative, differ markedly in structure. Gram-positive cell walls are simpler, consisting of a very thick layer of peptidoglycan (30–70% of dry weight). Teichoic acids are incorporated into this layer. The order from inside to outside is: Plasma Membrane → Thick Peptidoglycan Layer (with Teichoic Acids). Gram-negative cell walls are complex, featuring a thin layer of peptidoglycan (less than 10% of dry weight) situated within the periplasmic space. This is covered by an outer membrane, where the outer leaflet is composed of lipopolysaccharide (LPS). The order from inside to outside is: Plasma Membrane → Periplasmic Space (containing thin Peptidoglycan layer) → Outer Membrane (LPS/Phospholipid Bilayer).

What microorganisms utilize chitin, peptidoglycan, LPS, or cellulose in the cell wall, and how do these building blocks differ in structure and function?

Microorganisms utilize distinct building blocks for their cell walls: Peptidoglycan is a complex, rigid polymer unique to Bacteria and provides cell strength. Cellulose is a polymer used by plants, algae, and some lower fungi, functioning primarily for structural strength. Chitin is a strong, flexible polysaccharide utilized by most fungi (like yeast and mushrooms). LPS (Lipopolysaccharide) forms the outer surface of the outer membrane in Gram-negative bacteria; its Lipid A component acts as an endotoxin.

What are the differences in purpose/function for solid and liquid media?

Solid media (like agar plates) are essential for the isolation of microbes to create pure cultures and are also used for long-term storage. Liquid media (broth) are used when the goal is the rapid and large-scale production of microbial biomass.

What are the differences in purpose/function for a streak plate and a pour plate?

The streak plate technique is the standard method for obtaining a pure culture by physically diluting the sample across the agar surface until single colonies form. The pour plate method involves mixing a diluted sample directly into molten agar before it solidifies, allowing for the isolation of cells that grow within the medium, such as those sensitive to surface oxygen levels.

What are fastidious organisms, and what is one example?

Fastidious organisms are microbes that are unable to synthesize many necessary nutrients and consequently require complex diets for growth. An example of such an organism is Neisseria gonorrhoeae.

What is the purpose of deep-freezing?

The purpose of deep-freezing (temperatures between −70∘C and −95∘C) is to provide long-term storage for microbial cultures, often lasting many years. The extremely low temperature halts cell growth and metabolic processes, preventing detrimental effects like genetic mutations or loss of cell viability.

What is the separation between autotrophs, heterotrophs, chemotrophs, phototrophs, lithotrophs, and organotrophs? What kind of troph is a plant, animal, bacteria, and fungi?

Organisms are separated based on their nutritional strategy: Autotrophs derive their carbon from carbon dioxide (CO2), while Heterotrophs require preformed organic compounds for carbon. Phototrophs gain energy from light, whereas Chemotrophs gain energy from chemical compounds. Within chemotrophy, Organotrophs use organic molecules as their electron source, and Lithotrophs use inorganic molecules. Plants are typically Photoautotrophs, commonly using inorganic electrons (lithotrophs). Animals and Fungi are Chemoheterotrophs (organotrophs). Bacteria are the most diverse group, with representatives across all categories.

What is the difference between defined and undefined media? What are examples of when each would be used?

Defined media are synthetically prepared so that the exact chemical composition of every component is known. They are typically used when studying the specific nutritional requirements of an organism and contain simple chemicals like glucose or NaCl. Undefined media (or complex media) have an unknown composition because they contain components of variable makeup, such as yeast extract or blood. These media are cheaper and easier to prepare and are often used to cultivate fastidious organisms because they effortlessly supply all necessary complex nutrients.

What is the difference between selective, differential, and enrichment media? What are examples of when each would be used?

Selective media, differential media, and enrichment media are specialized tools microbiologists use to help grow or identify specific organisms from a mixed community. Selective media encourages the growth of a particular organism or group, usually by including components that suppress the growth of everything else; for instance, bismuth sulphite medium contains bismuth ions that inhibit Gram-positive bacteria, allowing the selection of Salmonella typhi. In contrast, differential media allows multiple organisms to grow, but they contain colored indicators or substances that enable visual differentiation between colonies based on specific metabolic activities, such as MacConkey agar distinguishing lactose fermenters (red) from non-fermenters (white/pale-pink). Enrichment media uses a selective approach to favor the growth of a desired organism that is initially present in very low numbers, such as using blood agar to encourage the isolation of streptococci.

What are the “macronutrients” vs. “micronutrients” required for cells?

Microorganisms require a variety of raw materials, classified generally as macronutrients and micronutrients. Macronutrients are elements required in large quantities for cellular structure and function, including the most abundant element, carbon, which forms the skeleton of all organic molecules. Other major macronutrients are nitrogen and phosphorus, necessary for synthesizing proteins, nucleic acids, and phospholipids, along with oxygen, hydrogen, sulphur, potassium, sodium, calcium, and iron. By contrast, micronutrients (or trace elements) are metal ions needed only in minute amounts, frequently functioning as cofactors crucial for regulating enzyme activity, such as copper, zinc, and molybdenum.

What are the differences between simple diffusion, facilitated diffusion, and active transport? Provide an example of when each would be used.

Cells employ several strategies for transporting substances across the selectively permeable plasma membrane. Simple diffusion is the easiest method, where very small molecules (like H2O or O2) or lipid-soluble nonpolar gases move passively down their concentration gradient until equilibrium is reached, requiring no energy. Facilitated diffusion handles larger, polar molecules (like sugars or amino acids) that cannot pass through the lipid barrier alone; these molecules use membrane-spanning transport proteins to diffuse down their concentration gradient, also requiring no energy, but relies on quickly metabolizing the molecule inside the cell to maintain the inward gradient. Finally, active transport is necessary when a cell needs to move substances against an unfavorable concentration gradient, often concentrating nutrients acquired from a dilute environment; this process always requires an input of cellular energy, typically derived from ATP hydrolysis, and utilizes specific transmembrane proteins.

What are the different kinds of oxygen requirements in aerobes and anaerobes for growth in media? Where would each kind of bacterium grow within a static culture growth?

Microbes are categorized based on their dependency on or sensitivity to oxygen (O2​) for growth, which dictates where they settle in a static culture tube. Obligate aerobes strictly require O2​ to act as their final electron acceptor, so they will grow densely only at the surface where O2​ diffuses into the medium. Obligate anaerobes cannot tolerate O2​ at all (as they lack enzymes to neutralize toxic oxygen radicals) and thus grow only in the deeper, anoxic zones of the culture. Facultative anaerobes can utilize oxygen if available for efficient aerobic respiration, but they can switch to anaerobic metabolism when O2​ is absent; consequently, they grow throughout the tube, but with the heaviest density near the oxygen-rich surface. Aerotolerant anaerobes do not use O2​ but possess defenses that allow them to survive its presence, leading to even, albeit modest, growth throughout the medium. Finally, microaerophiles require O2​ but only tolerate low concentrations (2%–10%), meaning they congregate in a specific narrow band just below the surface.

How are the different kinds of bacteria defined based on their temperature requirements for growth, and what is their optimal temperature? What is an example for each? (thermophiles/mesophiles/psychophiles/extremophiles)

Microorganisms are defined based on their specific temperature requirements for optimal growth, reflecting the thermal stability and efficiency of their cellular enzymes. Mesophiles (middle-loving) prefer moderate temperatures, growing optimally between 20∘C and 45∘C, a category that includes most human pathogens. Thermophiles (heat-loving) thrive at high temperatures, typically between 40∘C and 80∘C, with an optimum around 50∘C to 65∘C. Psychrophiles (cold-loving) are adapted to very low temperatures, capable of growth at 0∘C with optimal temperatures at 15∘C or below, such as some Archaea found in polar regions. Extremophiles (hyperthermophiles) represent the upper limit, thriving above 80∘C, with some having optimum temperatures exceeding 100∘C.

How does osmotic pressure impact cells, and what can happen to cells that become too hypo- or hypertonic?

Osmotic pressure refers to the force that controls water movement across a cell membrane, and changes in this pressure can severely impact cell integrity. When a cell is placed in a hypotonic solution (low solute concentration, often the case for bacteria in nature), water tends to flow into the cell; while the rigid bacterial cell wall usually prevents the cell from bursting, excessive internal pressure can still be damaging. Conversely, when a cell is placed in a hypertonic solution (high solute concentration), water flows out of the cell; this osmotic loss causes the plasma membrane to shrink away from the cell wall, a process called plasmolysis, which is often exploited when using high salt or sugar concentrations to preserve food against microbial attack.

What factors affect microbial growth (like pH)? How does this impact and/or stop microbial growth?

Factors such as pH (the concentration of hydrogen ions) strongly influence microbial growth, as most species are limited to a narrow tolerance range, generally around neutrality (pH 7). When the pH deviates significantly from an organism's optimal range, it profoundly impacts microbial viability because extreme acidity or alkalinity alters the ionization of charged groups on the amino acids that make up cellular proteins (especially metabolic enzymes). This alteration disrupts the three-dimensional structure of the proteins, leading to denaturation and a subsequent loss of the crucial catalytic function, ultimately halting or stopping microbial growth. Other key environmental factors that similarly affect growth and cause enzyme denaturation include temperature and solute concentration.

What is the typical bacterial growth curve? How does a diauxic growth curve differ?

The typical bacterial growth curve describes the increase in a microbial population over time in a closed vessel (batch culture), proceeding through four sequential phases: a lag phase of adaptation where no net growth occurs; an exponential (log) phase of rapid, predictable doubling; a stationary phase where growth halts and the rate of cell division equals the rate of cell death due to limited nutrients or waste accumulation; and finally, a death phase where viable cell numbers decline. A diauxic growth curve differs from this pattern because it results from the preferential metabolism of two different substrates present in the medium. The curve shows an initial exponential phase (using the preferred substrate, such as glucose), followed by a brief second lag phase (as the cell synthesizes new enzymes for the second substrate), and then a second exponential phase (using the less favored substrate), creating a characteristic biphasic curve.

How do direct microscopic examination, cell-sorting, viable cell counting, and turbidimetric (OD) methods differ for counting and monitoring cell growth? What functions do they serve in the real world?

Scientists monitor microbial population size using various techniques that serve specific real-world functions. Direct microscopic examination involves manually counting cells on a specialized slide (like a Petroff-Hauser chamber) of known volume, which is accurate for total cell numbers but cannot distinguish living from dead cells. Cell-sorting uses specialized equipment to automatically count cells passing through a detector, providing a rapid total count, but also cannot differentiate viability. Viable cell counting uses serial dilution and plating to estimate the concentration of Colony-Forming Units (CFUs), thus counting only the actively replicating (living) cells, which is vital for monitoring water quality or food contamination. Finally, turbidimetric (Optical Density or OD) methods provide a very fast, indirect measure of cell biomass by using a spectrophotometer to measure how much light the cloudy culture scatters, enabling instant monitoring of growth progression in liquid cultures.

What is metabolism, anabolism, and catabolism? How are they all connected to each other?

Metabolism is the term encompassing all the biochemical reactions that take place inside a living cell, composed of two interdependent parts: catabolism and anabolism. Catabolism involves the chemical breakdown of larger molecules (like nutrients), a process coupled to the release of chemical energy in the form of ATP and reducing power. Anabolism refers to the synthetic reactions where small molecules are built up into complex macromolecules like proteins and nucleic acids, which always requires an input of energy. Thus, the two are constantly linked: catabolism harvests energy from nutrient breakdown, and anabolism consumes that energy (ATP) to build and maintain the cell's structures.

What is the Entner-Doudoroff pathway? When and where is it used, and what are the substrates and final products?

The Entner-Doudoroff Pathway is an alternative route to glycolysis used by certain bacterial groups, notably pseudomonads (Gram-negative). This pathway, which occurs in the prokaryotic cytosol, oxidizes glucose (the substrate) to produce pyruvate and glyceraldehyde-3-phosphate, the latter of which then feeds directly into the lower part of the glycolysis pathway. The cell uses this pathway to achieve a net yield of one ATP, one NADH, and one NADPH per glucose molecule.

What is the pentose phosphate pathway? When and where is it used, and what are the substrates and final products?

The Pentose Phosphate Pathway (also known as the hexose monophosphate shunt) is a metabolic bypass that runs concurrently with glycolysis in the cytosol and is primarily utilized for anabolic (biosynthetic) functions, rather than just energy production. Starting typically with glucose-6-phosphate (the substrate), this pathway generates essential precursor molecules like ribose-5-phosphate for nucleotide synthesis and erythrose-4-phosphate for amino acid synthesis. It also supplies the cell with reducing power in the form of NADPH.

How is Oxidation-Reduction (Redox Potential) utilized in cell metabolism?

Oxidation-Reduction (Redox Potential) reactions are fundamental to cell metabolism because they facilitate energy flow. Metabolism involves transferring electrons from one molecule (oxidation, the loss of electrons) to another (reduction, the gain of electrons), a necessary link since the two processes are irrevocably coupled. The redox potential determines the affinity a substance has for electrons; by arranging electron carriers (like cytochromes) in a defined sequence based on increasingly positive redox potentials, cells utilize electron transport chains in respiration and photosynthesis. This electron flow releases energy gradually, which is captured to generate a proton motive force that drives ATP synthesis. Coenzymes like NAD+/NADH and FAD/FADH2​ shuttle electrons from energy-releasing reactions (like the TCA cycle) to the chain.

What are the differences in energy efficiency and uses for carbs, proteins, and lipids?

In terms of energy storage, nutrients vary widely, reflecting differences in their chemical structure. Lipids (fats) represent the richest and densest energy source because they contain a higher proportion of energy-storing Carbon-Hydrogen (C−H) and Carbon-Carbon (C−C) bonds compared to other macromolecules. Carbohydrates like glucose are the most common energy source, yielding the highest number of ATP molecules per molecule (up to 38 in prokaryotes) when completely oxidized. Carbohydrates enter catabolism primarily through glycolysis. Lipids are broken down through β-oxidation into Acetyl-CoA and glycerol, which feed into the TCA cycle and glycolysis, respectively, making them a rich caloric source. Proteins are generally the least preferred energy source but can be utilized when carbohydrates and lipids are scarce by breaking them into amino acids, which are then deaminated and channeled into the TCA cycle.

What is the Krebs (TCA) cycle? When and where is it used, and what are the substrates and final products?

The Krebs cycle, also known as the Tricarboxylic Acid (TCA) cycle or the citric acid cycle, is a series of chemical reactions designed to completely oxidize organic carbon molecules into carbon dioxide (CO2​). This process follows glycolysis in cellular respiration. In prokaryotes, it occurs right in the cytoplasm, while in eukaryotes, it takes place within the mitochondrial matrix. The primary substrate that enters the cycle is Acetyl-CoA, which is derived from the pyruvate produced during glycolysis. For each Acetyl-CoA molecule that completes the cycle, the main products generated for energy transfer are three molecules of NADH and one molecule of FADH2​, along with the removal of two carbon atoms as CO2​. The high-energy electrons stored in the NADH and FADH2​ molecules are subsequently used to generate a much larger supply of ATP in the final stage of respiration.

What is Glycolysis? When and where is it used, and what are the substrates and final products?

Glycolysis is the initial metabolic pathway used by nearly all living cells to break down sugars. This pathway, sometimes called the Embden-Meyerhof pathway, works regardless of whether oxygen is present. Its purpose is to split sugar molecules, which occurs in the cytoplasm of both prokaryotic and eukaryotic cells. The starting substrate is Glucose (a 6-carbon sugar), and the process requires an initial investment of two ATP molecules to kick off the reaction. After a series of ten linked reactions, the final organic product is two molecules of pyruvate (a 3-carbon compound). In terms of energy, glycolysis provides a small net yield of two ATP molecules and produces two molecules of NADH (which carries reducing power).

What are the differences between bacteriochlorophyll and chlorophyl? What is the absorbance spectrum of Chlorophyl A?

The primary difference between chlorophyll and bacteriochlorophyll lies in the type of photosynthesis they facilitate and the specific wavelengths of light they absorb. Chlorophyll A and B are the major pigments found in organisms performing oxygenic photosynthesis (like algae and plants). Bacteriochlorophylls are found in bacteria that perform anoxygenic photosynthesis, and they are adapted to absorb light at longer wavelengths (into the infrared spectrum), allowing them to utilize light that penetrates beyond the surface layers of water. The visible absorbance spectrum of Chlorophyll A shows that it primarily captures light in the red and blue regions of the visible spectrum, while it reflects or transmits light in the green region, which is why organisms containing this pigment typically appear green.

What are the differences between Glycolysis, Krebs, Entner-Doudoroff, and pentose phosphate pathways?

The four glucose metabolic pathways—Glycolysis, the Entner-Doudoroff Pathway (ED), the Pentose Phosphate Pathway (PPP), and the Krebs (TCA) Cycle—each serve distinct roles. Glycolysis is the widespread primary catabolic route that oxidizes glucose to pyruvate, netting two ATP and two NADH for energy. The ED pathway is an alternative catabolic route used by certain bacteria, like pseudomonads, yielding less ATP but also generating NADPH along with NADH. Unlike the other three, which are primarily related to energy harvesting, the PPP functions mainly as an anabolic (biosynthetic) bypass, generating crucial precursor molecules (like pentose sugars for nucleotides) and NADPH for reductive synthesis reactions. Finally, the Krebs Cycle takes the products of glycolysis (acetyl-CoA) and fully oxidizes them to CO2​, yielding minimal ATP directly but maximizing the production of energy-carrying molecules (NADH and FADH2​) to feed the final stage of respiration.

What is the Calvin cycle? When and where is it used, and what are the substrates and final products?

The Calvin cycle is the most common mechanism used by microbes to fix inorganic carbon (CO2​) into usable organic material, such as hexose sugars. This cycle, often called the "dark reactions" because it doesn't directly require light, is essential for all autotrophic organisms, including photosynthetic microbes and certain chemoautotrophs. The process takes place in the stroma of chloroplasts in eukaryotes or the cytoplasm/thylakoids of prokaryotes. The cycle consumes the chemical energy stored during the light reactions, utilizing ATP and NADPH. The enzyme Rubisco fixes the incoming CO2​ onto an existing 5-carbon sugar, and the output is ultimately glyceraldehyde 3-phosphate (G 3-P), which the cell converts into final products like glucose.

What are the different mechanisms to regulate cellular metabolism?

Cellular metabolism is constantly regulated to prevent wasteful or harmful synthesis, primarily by controlling the activity of key enzymes. This regulation happens through two main types of mechanisms: direct control of enzyme activity and indirect control at the genetic level. Direct control is typically seen in feedback (or end-product) inhibition, where the final product of a pathway physically binds to and temporarily shuts down an enzyme near the beginning of that same pathway. Indirect control involves altering the rate at which enzymes are synthesized (transcriptional control) through mechanisms like induction (switching genes on when a substrate is present, such as the lac operon) or repression (switching genes off when an end product is abundant, such as the trp operon).

What are the differences between NAD+ and NADP+, and what functions do they serve within a cell?

Regarding redox carriers, both NAD+ and NADP+ are coenzymes that shuttle electrons. However, NAD+/NADH is generally employed in catabolic reactions (breaking molecules down to harvest energy), while NADP+/NADPH is primarily utilized in anabolic reactions (building complex molecules, requiring reducing power).

How are aerobic respiration, anaerobic respiration, and fermentation connected and how do they differ?

Aerobic respiration, anaerobic respiration, and fermentation are all energy-harvesting strategies that start with glycolysis and serve the universal purpose of oxidizing NADH back to NAD+ so that glycolysis can continue. Respiration (both aerobic and anaerobic) is characterized by the transfer of electrons via an electron transport chain (ETC), generating energy efficiently through oxidative phosphorylation. The difference between the two lies in the terminal electron acceptor: aerobic respiration requires oxygen (O2​) and yields the maximum ATP (up to 38 molecules per glucose), while anaerobic respiration uses other inorganic molecules (like nitrate or sulfate) and yields less ATP. In contrast, fermentation does not utilize an ETC or oxygen and generates minimal energy solely via substrate-level phosphorylation in the cytoplasm (just two ATP per glucose). Fermentation quickly re-oxidizes NADH to NAD+ to keep glycolysis cycling, often creating organic end products like lactic acid or ethanol.

What is the electron transport chain? When and where is it used, and what are the substrates and final products? How does ATP Synthase use chemiosmosis to produce ATP?

The electron transport chain (ETC) is a series of molecules (like flavoproteins and cytochromes) embedded in a membrane that transfer electrons gradually from high-energy carriers (NADH and FADH2​) to a final electron acceptor, typically oxygen (O2​) in aerobic systems. In prokaryotes, the ETC is located on the plasma membrane, while in eukaryotes, it is confined to the inner mitochondrial membrane. The process ultimately converts O2​ into water (H2​O). The critical function of the ETC is to release energy in controlled steps, which is used to pump protons (H+) across the membrane, creating an electrochemical gradient called the proton motive force. The ATP synthase enzyme then uses this proton motive force in a process called chemiosmosis: H+ ions flow back through the specialized ATP synthase channel, and the energy released by this movement is harnessed to convert ADP into ATP.

What is photosynthesis? When and where is it used within a cell, and what are the substrates and final products? What are the differences between oxygenic and anoxygenic photosynthesis?

Photosynthesis is the crucial metabolic process where light energy is captured by pigments, primarily chlorophyll, and converted into usable chemical energy (ATP) to drive the synthesis of carbohydrates by reducing carbon dioxide. In photosynthetic organisms, the light-harvesting pigments are housed on internal thylakoid membranes. The overall substrates for photosynthesis are typically CO2​ and water (H2​O), yielding carbohydrate (C6​H12​O6​). Photosynthesis is divided into two types based on its inputs and outputs: Oxygenic photosynthesis (performed by plants, algae, and cyanobacteria) uses H2​O as the electron donor and produces oxygen (O2​). Anoxygenic photosynthesis (performed by purple and green bacteria) uses other electron donors such as hydrogen sulfide (H2​S) and therefore does not produce O2​.

What are the functions and differences of Photosystem I and Photosystem II?

The photosynthetic process in oxygenic organisms requires two key pigment-protein complexes: Photosystem I (P700​) and Photosystem II (P680​), which work sequentially in the "Z" scheme. Photosystem II is where the process begins, as it is specialized to oxidize (split) water, generating O2​ and releasing electrons. These electrons travel through an electron transport chain, generating ATP before being donated to Photosystem I. Photosystem I is responsible for reducing the coenzyme NADP+ to NADPH. Anoxygenic photosynthetic bacteria, however, typically employ only a single photosystem that resembles either PS I or PS II.

What is photophosphorylation? When and where is it used within a cell, and what are the substrates and final products? What are the differences between cyclic and non-cyclic photophosphorylation?

Photophosphorylation is simply the synthesis of ATP using energy originally derived from light. This crucial energy-generating step occurs on the thylakoid membranes via a chemiosmotic mechanism driven by the flow of electrons through carriers. There are two methods of photophosphorylation: Non-cyclic photophosphorylation utilizes both Photosystem I and Photosystem II, drawing electrons from water and donating them ultimately to NADP+. This process yields ATP, NADPH, and O2​. Cyclic photophosphorylation uses only Photosystem I; its electrons cycle back to the P700​ chlorophyll, generating a proton motive force and resulting only in the production of ATP. This cyclic route does not require water splitting and produces no O2​ or NADPH.

What are the most defining differences between Archaea, Eukarya, and Bacteria? Genetic material, presence of plasmids/histones/introns, ribosome structure, membrane fatty acids, cell wall, energy generation location, organelles, etc.

The three domains of life—Archaea, Eukarya, and Bacteria— are characterized by fundamental cellular and genetic differences. Eukarya are the most structurally complex, possessing a true nucleus containing multiple linear chromosomes associated with histone proteins, and having internal membrane-bound organelles like mitochondria. Archaea and Bacteria are prokaryotes (lacking a nucleus/organelles) and typically have a single circular dsDNA chromosome. However, they diverge significantly: Bacteria have cell walls usually made of peptidoglycan, use ester-linked fatty acids in their membranes, and lack histones. Archaea lack peptidoglycan, use unique ether-linked, branched fatty acids in their membranes, and do possess histones. Both prokaryotic domains use smaller 70S ribosomes, while Eukarya use 80S ribosomes.

What is 16S rRNA and where is it within a cell? How do Phylogenetic classifications function using this?

16S rRNA is a nucleic acid sequence found specifically within the small subunit (30S) of the prokaryotic 70S ribosome. It serves an essential, common function in protein synthesis in all organisms. In phylogenetic classifications, the gene sequence of the 16S rRNA is used because, while the sequence is largely conserved, accumulated minor differences over evolutionary time can be accurately measured. The degree of dissimilarity in the 16S rRNA sequences between two organisms provides a reliable estimate of their evolutionary distance, making it the central tool for establishing the phylogenetic tree that defines the three domains of life: Archaea, Bacteria, and Eukarya.

What key bacterial species belong to the phylum Proteobacteria? What are the defining characteristics of and separations within Proteobacteria?

The phylum Proteobacteria is the largest and most diverse single phylum within the Bacteria domain, containing approximately one-third of all known bacterial species. This phylum is categorized based on phylogenetic history, specifically 16S ribosomal RNA sequencing, which suggests all members evolved from a common ancestor, likely photosynthetic. However, few members still possess this ability. Because classification is based on molecular relatedness rather than shared traits, the group, which includes many important Gram-negative bacteria, is incredibly diverse in morphology and metabolism, leading to its name (after the Greek god Proteus who could assume many forms). For classification purposes, it is separated into six major classes (α,β,γ,δ,ϵ,ζ). Key species belong to classes like Gammaproteobacteria (e.g., Escherichia, Shigella, Salmonella, Yersinia (plague), and Pseudomonas), Alphaproteobacteria (e.g., Rhizobium and Agrobacterium), and Betaproteobacteria (e.g., Neisseria).

What key bacterial species belong to the phylum Cyanobacteria? What are the defining characteristics of and separations within Cyanobacteria?

The Cyanobacteria phylum, previously and mistakenly known as blue-green algae, contains Gram-negative prokaryotes. Their defining characteristic is that they are the only group of prokaryotes capable of oxygenic photosynthesis (a process shared with eukaryotes like plants and algae). Since they lack chloroplasts, they contain specialized thylakoid membranes within the cytoplasm, which serve as the location for light-gathering pigments and electron transfer processes. The Cyanobacteria are thought to have been responsible for the gradual increase of oxygen in the Earth’s ancient atmosphere. Furthermore, many members of this phylum can perform nitrogen fixation, reducing atmospheric nitrogen (N2​) to ammonia (NH4+​), often using specialized cells called heterocysts to protect the sensitive nitrogenase enzyme from oxygen. Representative genera include Oscillatoria and Anabaena.

What key bacterial species belong to the phylum Bacteroidetes? What are the defining characteristics of and separations within Bacteroidetes?

The Bacteroidetes phylum includes diverse species that are phylogenetically related but lack a single unifying phenotypic trait or characteristic. One of the key genera is Bacteroides, which are obligate anaerobes found most prominently in the human gut. Within the gut, Bacteroides ferments undigested food, producing compounds like acetate and lactate. The phylum also includes organisms like Flavobacterium

What key bacterial species belong to the phylum Firmicutes? What are the defining characteristics of and separations within Firmicutes?

The Firmicutes phylum is composed of Gram-positive bacteria typically characterized by a low GC content (guanine and cytosine residues) in their DNA. Most members are chemoheterotrophs. The phylum contains important groupings defined by their physiology: genera that produce endospores (like the potent exotoxin producers Clostridium and Bacillus) and non-spore-forming groups (like the lactic acid bacteria, including Streptococcus and Lactobacillus, and Staphylococcus). Clostridium species are obligate anaerobes that rely solely on fermentation (lacking an electron transport system), while Staphylococcus species are facultative anaerobes known for their resistance to drying and salt tolerance, allowing them to colonize the human skin.

What key bacterial species belong to the phylum Actinobacteria? What are the defining characteristics of and separations within Actinobacteria?

The Actinobacteria phylum consists of high GC content Gram-positive bacteria. Many species are referred to as actinomycetes because they are aerobic filamentous bacteria that form branching mycelia that look similar to fungi. This group is ecologically important as decomposers in soil. Crucially, they are a major source of clinically useful antibiotics, including streptomycin and erythromycin. Key genera include Streptomyces and pathogenic species such as Mycobacterium (tuberculosis) and Corynebacterium (diphtheria).

What are pathogenicity islands, and how do they function? What is an example?

Pathogenicity Islands (PAIs) are genomic regions that pathogens acquire via horizontal gene transfer (HGT). These regions act as clusters of genes that contain various virulence factors, such as toxins, adhesins, capsules, or secretion systems, allowing the microbe to better colonize and harm a host. A PAI may be located on the bacterial chromosome, a plasmid, or even within a bacteriophage genome. PAIs possess genes that enable them to insert into the bacterial DNA, frequently targeting transfer RNA (tRNA) genes. The most frequent way PAIs are transferred between bacteria is via conjugative plasmids, which can rapidly confer virulence to a previously harmless bacterium. For example, the diphtheria toxin gene is carried on the tox gene, which is passed to the Corynebacterium diphtheriae bacterium via infection by a bacteriophage.

What role do Type IV pili, Type III Secretion Systems, and Type VI Secretion Systems play in bacterial quorum sensing, bacterial interactions, & biofilms? What cells possess these mechanisms?

Type IV pili, Type III Secretion Systems (T3SS), and Type VI Secretion Systems (T6SS) all play roles in bacterial interactions, biofilms, and quorum sensing-regulated behaviors. Type IV pili primarily enable bacteria, such as P. aeruginosa, to attach to surfaces and perform twitch motility (a crawling movement). At low cell densities, they are essential for the initial, reversible attachment phase of biofilm formation and for resisting flushing. The T3SS is a common injection system that functions like a specialized syringe anchored in the cell wall, used to deliver bacterial effector proteins directly into a host cell to manipulate its function. T3SS expression is often activated once a bacterial population has reached a high cell density (a quorum). The T6SS is often utilized for bacterial warfare, injecting antibacterial effector molecules into competitor bacteria, though it can also be used to inject effectors into eukaryotic cells. For instance, Vibrio cholerae uses its T6SS to kill competitors and lyse non-immune cells, releasing DNA that subsequently promotes HGT via transformation.

How do single and multicellular behavior differ within a cell depending on its environment?

Bacterial behavior is a dynamic response to population size, regulated by quorum sensing. At low cell densities, the population defaults to single-cell behavior, where cells exhibit traits like high motility (flagella) and reversible attachment (Type IV pili) to successfully colonize the most favorable niche and avoid detection by the host immune system. However, in this state, virulence factors are used less effectively and the population is easier to clear. When the population reaches a high cell density (a quorum), multicellular behavior is activated. The cells irreversibly attach, form protective biofilms, and simultaneously express virulence factors to overwhelm the host. This coordinated assault allows for better stress resistance but increases competition for nutrients and makes the population more detectable.

What are the mechanisms and roles within Bacterial Quorum Sensing? What are autoinducers used for with quorum sensing?

The central role of Bacterial Quorum Sensing is to determine population size using autoinducers (signaling molecules) and then modulate gene expression to execute coordinated behaviors. Autoinducers are produced and excreted by individual cells and bind to receptors either in the cytoplasm or on the surface of the bacteria. Once these molecules reach a critical threshold level (a quorum), the autoinducer/receptor complex binds to DNA promoters, activating quorum sensing genes and initiating the shift to multicellular behavior. Autoinducers differ based on the target: Gram-negative bacteria typically use AHLs (acyl-homoserine lactones, AI-1 family) that freely diffuse and bind to intracellular receptors, while Gram-positive bacteria use short oligopeptides that are actively exported and bind to extracellular receptors, initiating a phosphorylation cascade. Additionally, AI-2 family autoinducers facilitate interspecies communication.

What are the stages of formation, mechanisms, and different roles within biofilm formation? How does quorum sensing play a role in the biofilm formation?

Biofilm formation is a key process enabled by quorum sensing, which allows bacteria to transition from single cells to a resistant, structured, multicellular community. The process begins with single cells performing reversible attachment via structures like Type IV pili. Once the population reaches a high density (a quorum), quorum sensing genes are activated, triggering irreversible attachment and the production of a protective polysaccharide matrix known as the glycocalyx (EPS). The mature biofilm, which is highly resistant to external stresses such as antibiotics and phagocytosis, forms three-dimensional structures with internal channels to facilitate the passage of water, nutrients, and oxygen. When local nutrients are depleted, quorum sensing reverses the gene expression, turning off adhesin genes and turning on flagella genes, allowing cells to disperse and colonize new sites.

How does yeast differ from multicellular forms of fungi? What phylum does yeast belong to?

Yeast are the unicellular form of fungi, reproducing primarily by budding (a protuberance that pinches off) or fission. In contrast, the multicellular forms, such as molds or mushrooms, grow as long, thin filaments called hyphae that aggregate into a network known as a mycelium. Some fungi are dimorphic, meaning they can switch between the yeast and mycelial forms, often in response to temperature or nutrient changes. Yeast belongs to the phylum Ascomycota.

What are the differences between endospores with bacteria and fungal spores with fungi?

The fundamental difference between bacterial endospores and fungal spores lies in their function and resistance: Endospores (produced by Bacillus and Clostridium) are dormant survival forms of the bacterial cell that are highly resistant to extreme environmental stresses, including high heat (surviving boiling at 100∘C for hours). They must be destroyed by wet heat at 121∘C. In contrast, fungal spores (produced by multicellular fungi) are non-motile reproductive cells used for dispersal (e.g., conidia, ascospores). While they possess resistance to environmental stress, they are not as resistant as endospores.

What key fungi species belong to the phylum Ascomycota? What are the defining characteristics of this phylum?

The Ascomycota phylum, often called the "sac" fungi, includes yeasts, molds, and truffles. The defining characteristic is their mode of sexual reproduction, which results in the production of haploid ascospores inside a specialized sac-like structure called an ascus. They reproduce asexually under favorable conditions by producing airborne asexual spores called conidia on specialized hyphae called conidiophores. Key species include yeasts like Saccharomyces cerevisiae, as well as many fungi involved in forming lichens.

What key fungi species belong to the phylum Glomeromycota? What are the defining characteristics of this phylum?

The Glomeromycota phylum is a small group characterized by having coenocytic hyphae (containing multiple nuclei without cross-walls or septa). They are ecologically important because they form internal mutualistic associations (mycorrhizae) with the roots of plants. Specifically, fungi in this phylum are the exclusive providers of arbuscular mycorrhizal fungi (AMF), characterized by highly branched structures (arbuscules) that penetrate the plant root cells to facilitate nutrient exchange.

What key fungi species belong to the phylum Basidiomycota? What are the defining characteristics of this phylum?

The phylum Basidiomycota, often called the "club fungi," is a large group containing approximately 30,000 species, including true mushrooms, toadstools, puffballs, and bracket fungi. They play a significant economic role in breaking down wood and other plant materials. Their defining characteristic is the production of basidiospores on club-shaped structures called basidia. Sexual reproduction often results in a striking aerial structure known as the basidiocarp (the mushroom itself). They mostly reproduce sexually, involving the fusion of compatible haploid hyphae to form a dikaryotic mycelium. A unique feature to ensure genetic consistency is the clamp connection. Asexual reproduction occurs much less frequently than in Ascomycota, usually via conidia or fragmentation of hyphae.

How does fungal nutrition differ between phyla? What are all the methods fungi can use to take in nutrients?

Fungal nutrition generally involves a chemoheterotrophic, absorptive mode of life. Most fungi are saprobes, meaning they obtain nutrients from decaying matter by secreting enzymes extracellularly to break down complex molecules into simpler forms that the hyphae can absorb. They typically store carbohydrates as glycogen. While most fungi are aerobic, some yeasts can function as facultative anaerobes. Different phyla exhibit different metabolic requirements; for example, members of the Neocallimastigomycota are a small group of anaerobic fungi found in the rumen of herbivores.

What is the phylogenetic history of the Protista? How did they come to exist?

The Protista are a highly diverse group of eukaryotic organisms that do not fit into the animal, plant, or fungal kingdoms. Traditionally, they were classified based on shared common traits or physical features, leading to an "artificial division". Molecular studies (phylogenetics) have revealed that some members of the Protista are only very distantly related to each other, making the traditional grouping taxonomically unsatisfactory. For example, organisms traditionally grouped as "algae" and "protozoa" may be phylogenetically related, suggesting that classification based solely on features like chloroplasts was misleading. The accepted three-domain system of life places Protists within the Eukarya domain.

What are the traits all protists have in common?

The traits that all organisms traditionally classified as Protists have in common are that they are all eukaryotes and most are single-celled. Some Protists may also exist in multicellular forms.

What defines the algae grouping of protists? What key features do they have?

The algae grouping of protists is an artificial division not based on phylogenetics. Key features include that they are all eukaryotes, often come in multicellular forms, possess the pigment chlorophyll (a, b, c, or d), perform oxygenic photosynthesis, and are autotrophs that use CO2​ or bicarbonate (HCO3−​) as their carbon source. With the exception of one group, most algae have a cellulose cell wall.

What defines the protozoan grouping of protists? What key features do they have?

The protozoan grouping of protists is an artificial division meaning "first animal" (though they were not). Most are heterotrophs that engulf food. They typically reside in aquatic systems or moist soil. A characteristic feature is the contractile vacuole, which controls osmotic pressure by pumping excess water out of the cell.

What defines the water mold (oomycetes) grouping of protists? What key features do they have?

The water mold (oomycetes) grouping of protists is an artificial division that resembles true fungi in gross structure (having hyphae). However, water molds are not related to fungi; instead, DNA analysis suggests they are related to diatoms and brown algae. Their cell walls are composed of cellulose. They are significant decomposers in freshwater ecosystems and some are parasites.

What defines the slime mold grouping of protists? What key features do they have?

The slime mold grouping of protists is also an artificial division. Plasmodial slime molds start as spores that germinate into a haploid cell, which can fuse into a diploid cell. The nucleus of the diploid cell then divides repeatedly without cell division, creating a plasmodium (a single membrane-bound mass of cytoplasm with multiple nuclei) that can move like an amoeba and phagocytose rotting vegetation. Cellular slime molds start as haploid cells that, when nutrients are limited, aggregate into slug-like blobs called pseudoplasmodia, which are composed of multiple cells. Both types develop fruiting bodies to produce haploid spores for reproduction.

Are viruses living organisms or nonliving? How is this defined? What is the typical size of a virus?

Viruses are not considered strictly living because they do not meet the definition of life; they are not cells, are incapable of metabolism, and do not increase in size as individuals. However, they are not quite non-living, as they show life-like replication once inside a host. Outside of a cell, viruses are simply inert chemical structures. The typical size of a virus is between 20 nm and 300 nm, though some can be larger.

What is the Baltimore system for virus classification, and what are the 7 groupings for viruses?

The Baltimore system for virus classification orders viruses based on the strategies they use for mRNA production. This system results in seven major groupings:

1. Group I: dsDNA viruses

2. Group II: ssDNA viruses

3. Group III: dsRNA viruses

4. Group IV: (+) sense ssRNA viruses

5. Group V: (-) sense ssRNA viruses

6. Group VI: Single-stranded (+) sense RNA with DNA intermediate

7. Group VII: Double-stranded DNA with RNA intermediate.

How are viruses cultivated for studying? How does this differ between plant, animal, and bacteriophage viruses?

Viruses are obligate intracellular parasites and therefore need an appropriate host cell to replicate, which presents special challenges for cultivation. For bacteriophages, they are grown in culture with their bacterial hosts, where successful propagation results in clearing of the culture's turbidity (lysis). Their quantity (titer) is measured using a plaque assay, where plaques (clear zones) form in a lawn of bacteria, and each plaque is assumed to result from a single viral particle (plaque-forming unit, pfu). Animal viruses can be cultivated using fertilized chicken eggs (embryos and membranes) or cell culture (monolayers in tissue culture flasks). Plant viruses are grown by rubbing the virus along with a mild abrasive onto the surface of a leaf to create a minor wound, allowing the virus to bypass the cell wall.

What is the function of reverse transcriptase and why is it important?

The function of reverse transcriptase (also called RNA-dependent DNA polymerase) is to convert an RNA template into DNA. This is important because it is a startling exception to the central dogma of molecular biology (that information flows from DNA to RNA to protein). This enzyme is found in retroviruses and hepadnaviruses, allowing them to integrate their genetic material into the host's genome.

What are prions? Why are they not considered a virus? Are they considered to be living organisms?

Prions are infectious agents composed solely of protein, first discovered in the 1980s. They are not considered a virus because they contain no nucleic acid. Prions are thought to be mutated versions of normal animal proteins that cause the normal version to refold into the mutant, self-replicating form. They cause several neurodegenerative disorders in mammals, such as scrapie (sheep), BSE ("mad cow disease"), and Creutzfeldt-Jakob disease (humans). They are generally not considered to be living organisms in the conventional sense.

What are viroids? Why are they not considered a virus? Are they considered to be living organisms?

Viroids are pathogens of plants that are many times smaller than the smallest virus. They consist only of a small circle of single-stranded RNA (ssRNA) and lack a protein coat. They are not considered a virus because they are composed only of nucleic acid (ssRNA) and do not code for protein product. Viroids are not considered to be living organisms.

Describe the fundamental components that form a complete viral particle (virion).

A complete viral particle, or virion, has a very simple structure, fundamentally consisting of a core of nucleic acid (RNA or DNA) protected by a protein coat, called a capsid. This combination is known as the nucleocapsid. In some virus types (most commonly animal viruses), the nucleocapsid is further encased by a membranous envelope that is partially derived from the host cell. This envelope often contains spikes (virus-derived proteins) that project from the surface and aid in attachment or penetration of the host cell.

Describe the subunits that form the viral protein coat (capsid), and list the two primary symmetrical arrangements they adopt. Explain the main protective role that the capsid serves, particularly in non-enveloped viruses.

The viral protein coat, known as the capsid, is the outermost layer of non-enveloped viruses, and it is built from repeating subunits called capsomers. These capsomers arrange symmetrically to form two main shapes: the icosahedral structure, which is a three-dimensional shape with 20 triangular faces, or the helical structure, which is a rod-like helix or tube. The primary protective role of the capsid, especially in non-enveloped viruses, is to shield the inner nucleic acid core from damaging environmental factors such as UV light and desiccation, as well as the acid and degradative enzymes found in places like the gastrointestinal tract.

Describe the origin of the lipid bilayer in an enveloped virus and the functional role played by the associated virus-derived protein spikes.

In an enveloped virus, the outer lipid bilayer layer (the envelope) is derived from the nuclear or cytoplasmic membrane of the previous host cell. This envelope also contains proteins, called spikes, which are derived from the viral genome itself, and project outward from the surface. The spikes are instrumental in allowing the virus to attach to and penetrate the host cell during the infection process.

Describe the four different combinations of nucleic acid type and strandedness that can constitute a viral genome.

Viral genetic material is highly diverse, categorized by four combinations of nucleic acid type and strandedness. The genome can be composed of either DNA or RNA,, and either of these can be single-stranded (ss) or double-stranded (ds). Therefore, the four major combinations of viral genomes are dsDNA, ssDNA, dsRNA, and ssRNA.

Explain why a multipartite viral genome, where segments are packaged in separate particles, requires multiple virions to successfully infect and initiate the replication cycle in a single host cell.

In cases where a virus has a multipartite genome, its genetic information is segmented into pieces that are packaged into separate viral particles. To successfully initiate the replication cycle inside a host cell, multiple virions must co-infect the same cell to ensure that all segments are present and capable of complementing each other to provide the complete genetic blueprint.

Describe the key structural components of a complex T-even bacteriophage, such as T4

A complex T-even bacteriophage, such as T4, has a distinct head plus tail structure. The top structure is an icosahedral head that encapsulates the double-stranded DNA genome. Connected below the head are the collar and the contractile sheath. At the base of the tail are the base plate and several tail fibres.

Outline the essential steps, in order, that all viruses must complete to successfully replicate within a host cell. Describe the events that occur during the latent period of a viral infection, distinguishing between the eclipse period and the period when virions are released.

All viruses must successfully complete a general sequence of events to replicate within a host cell, beginning with Attachment, followed by Penetration, Replication, Assembly, and finally Release,. The latent period is the total time elapsed between the initial attachment of the viral particle and the subsequent release of newly synthesized phages. The initial phase of this latent period is the eclipse period, during which the cell contains components of the phage but no complete viral particles are present inside the host. The subsequent synthesis and release of new viral particles conclude the latent period.

Distinguish between a viral infection following a lytic cycle versus a lysogenic cycle.

A viral infection can follow one of two paths: the lytic cycle or the lysogenic cycle. In the lytic cycle, often carried out by virulent phages, the infection culminates in the lysis (bursting) and death of the host cell as new viral particles are released. In contrast, the lysogenic cycle, typical of temperate phages, involves the phage DNA (called a prophage) becoming integrated into the host's genome, where it is replicated along with the host chromosome without immediately causing harm. Repressor proteins maintain this lysogenic state because they are encoded by the phage and function to prevent most of the other phage genes from being transcribed,. However, the lysogenic state is threatened when the host experiences stress, such as exposure to a chemical mutagen; this environmental stressor can lead to a shift to the lytic cycle because it causes the inactivation of the repressor protein, allowing the phage DNA to be excised from the chromosome, thereby initiating the destruction of the host cell.

Describe the mechanism by which repressor proteins encoded by a temperate phage (like lambda) maintain the host cell in the lysogenic state. If a bacterial population is suddenly exposed to a chemical mutagen, explain why this environmental stressor might lead to a dramatic shift from lysogeny to the lytic cycle in temperate phages

The lysogenic state, seen in temperate phages like lambda (λ), is actively maintained by repressor proteins encoded by the phage itself. These proteins function to prevent the transcription of most of the other phage genes, ensuring that the phage DNA, known as a prophage, remains silently integrated within the host bacterium's chromosome and is faithfully replicated along with it. If a bacterial population is suddenly exposed to a high level of environmental stress, such as a chemical mutagen, this stress can threaten the host cell's survival and acts as a signal to trigger the end of the lysogenic cycle. This environmental stress leads to the inactivation of the repressor protein. Once the repressor is removed, the prophage DNA is excised from the chromosome, adopts a circular form in the cytoplasm, and immediately begins the destructive lytic cycle that culminates in the host cell's lysis and the release of new phages.

Explain why double-stranded RNA viruses require the activity of both their positive and negative RNA strands for protein synthesis and replication

Finally, double-stranded RNA (dsRNA) viruses are all segmented, meaning they rely on both strands for function: the (+) strand is transcribed into mRNAs for protein synthesis, and the (−) strand is required as a template for the synthesis of the new dsRNA genome itself.

Explain why (+) sense single-stranded RNA viral genomes are considered infectious on their own, while (-) sense ssRNA genomes require an associated polymerase to initiate replication

The structure of viral RNA dictates how replication is initiated: (+) sense single-stranded RNA (ssRNA) viral genomes are immediately infectious because they can act directly as messenger RNA (mRNA), ready to be translated by the host ribosomes into viral proteins. Conversely, (−) sense ssRNA genomes cannot function directly as mRNA, and must first be converted into their complementary (+) sense strand by a virally encoded RNA polymerase (which must be carried within the capsid).

Given a newly discovered antiviral drug that specifically blocks the synthesis of the complementary (-) sense RNA strand in a (+) sense ssRNA virus, describe the immediate consequences for the viral replication cycle

If a newly discovered antiviral drug were to specifically block the synthesis of the complementary (-) sense RNA strand in a (+) sense ssRNA virus, the immediate consequence would be the cessation of viral genome replication, as the complementary (-) sense strand is required to act as the template for creating new genomic (+) sense RNA molecules. Furthermore, if an inhibitor successfully blocked the formation of subviral aggregates in a double-stranded RNA (dsRNA) virus, the final assembly stage would be blocked because these aggregates, composed of translated viral proteins, function as the necessary template structure for synthesizing the new dsRNA genome.

Describe the functional role of the viral RNA-dependent RNA polymerase in the replication cycle of a negative (-) sense single-stranded RNA virus

The functional role of the RNA-dependent RNA polymerase is critical for the replication of a negative (-) sense single-stranded RNA virus because this genomic RNA cannot be read directly by host ribosomes. This polymerase must be virally encoded and carried within the viral capsid, and its job is to use the incoming genomic (-) sense RNA as a template to create a complementary (+) sense RNA strand. The resulting (+) sense strand then serves two purposes: acting as the necessary messenger RNA (mRNA) for viral protein synthesis and serving as a template for synthesizing more genomic (-) sense RNA

Describe the role of reverse transcriptase in the retroviral replication cycle, including the intermediate hybrid molecule formed.

The central role of reverse transcriptase in the retroviral replication cycle is to violate the historical statement of the central dogma of molecular biology—that genetic information flows strictly from DNA to RNA to protein—by allowing information to flow backward. This enzyme, which is an RNA-dependent DNA polymerase, uses the retroviral RNA genome as a template to synthesize a complementary strand of DNA. This process creates an initial RNA/DNA hybrid molecule as an intermediate. After the RNA strand is degraded, the reverse transcriptase synthesizes the second DNA strand, resulting in double-stranded proviral DNA that is then integrated into the host's chromosome.

Describe the primary physical barrier a plant virus must overcome during penetration and the common biological method used to bypass this barrier

The primary physical barrier a plant virus must overcome during penetration is the plant's rigid cellulose cell wall and associated cuticle. Plant viruses typically bypass this barrier via mechanical damage, often caused biologically by insects whose mouthparts puncture the wall to transmit the virus. Given that most animal viruses spread systemically throughout the host via the bloodstream, plant viruses utilize the plasmodesmata—which are naturally occurring cytoplasmic strands forming continuous pores between neighboring plant cells—to spread systemically throughout the host plant once the initial cell barrier is overcome.

Describe the semi-conservative mechanism by which genetic material is copied during replication

Genetic material is copied via semi-conservative replication, a process where the resulting daughter DNA molecule is composed of one parental strand and one newly synthesized strand. During this process, the parental double helix unwinds, allowing each original strand to act as a template against which a new complementary strand is synthesized. This synthesis relies on the strict rules of base-pairing to ensure faithful copying of the genetic sequence.

Differentiate between the roles of DNA Polymerase I and DNA Polymerase III in synthesizing new DNA strands and ensuring accuracy.

DNA Polymerase III is the main enzyme responsible for synthesizing the new DNA strand during replication by adding complementary nucleotides in the 5′ to 3′ direction, extending the chain from an existing 3′-OH group. DNA Polymerase I has a crucial clean-up role: it removes the short RNA primers that initiate DNA synthesis and replaces those RNA bases with DNA nucleotides. Importantly, both DNA Polymerase I and DNA Polymerase III ensure accuracy by performing a proofreading activity, allowing them to cut out incorrect nucleotides and replace them with the correct ones, resulting in a very low final error rate.

Explain the roles of accessory enzymes, including helicase and DNA topoisomerase, at the replication fork.

The replication fork is the Y-shaped site where DNA unwinding and synthesis occur. Accessory enzymes are necessary to prepare the DNA template for replication. Helicase acts like a zipper, causing the two strands of the DNA double helix to separate. As the double helix is opened up, it creates increased tension or supercoiling elsewhere in the molecule; this stress is relieved by the enzyme DNA topoisomerase (also known as DNA gyrase). Other accessory proteins, like single-stranded DNA binding proteins (SSB), help prevent the separated strands from immediately rejoining.

Describe how the requirement that DNA Polymerase III only synthesizes DNA in the 5′ to 3′ direction leads to the distinction between the leading and lagging strands.

The structure of DNA Polymerase III dictates that it can only synthesize new DNA by extending an existing strand in the 5′ to 3′ direction, because it requires a free 3′-OH group to attach new nucleotides. Since the two strands of the DNA template run in opposite polarity, this fundamental rule creates a distinction between them. The leading strand can be copied continuously in the same direction that the replication fork moves. However, the lagging strand runs in the opposite direction and must be synthesized discontinuously in short pieces called Okazaki fragments, where the polymerase works backward from the fork.

List the differences in DNA replication initiation and overall processivity between prokaryotic (e.g., E. coli) and eukaryotic linear chromosomes.

DNA replication differs between prokaryotes and eukaryotes largely due to their chromosomal structure and size. Prokaryotes, such as E. coli, typically have a single, circular chromosome and initiate replication at a single origin of replication sequence. Replication proceeds bidirectionally, creating one replication bubble that moves along the chromosome until completion. Eukaryotes, by contrast, possess multiple, linear chromosomes. Due to the much larger genome size and slower replication rates, replication initiates at numerous replication forks that are active simultaneously along each chromosome, forming many replication bubbles that merge to cover the entire length.

Compare and contrast the challenges faced by prokaryotic and eukaryotic cells in completing DNA replication, focusing on the concepts of circular versus linear chromosomes and single versus multiple origins of replication.

Prokaryotic cells typically manage a single, circular chromosome that is replicated efficiently by starting at a single origin and moving bidirectionally until the forks meet. The challenge here includes managing the supercoiling created by unwinding that large circular molecule. Eukaryotic cells face the difficulty of replicating multiple linear chromosomes which are generally much larger. To overcome the sheer length and slower speed of replication, eukaryotes use multiple origins on each linear chromosome, leading to numerous replication bubbles that merge and ultimately ensure that the entirety of the long linear molecules is copied.

A new chemical is introduced that specifically degrades RNA primers. Explain how this chemical would inhibit DNA replication on the leading versus the lagging strand.

The synthesis of new DNA strands is initiated by a short segment of RNA called an RNA primer, which provides the necessary 3′-OH starting point for DNA polymerase. On the leading strand, only one primer is needed to begin continuous synthesis, so replication would be completely blocked from starting. The effect on the lagging strand would be much more severe, as it requires multiple RNA primers, one for the initiation of every short Okazaki fragment. Replication on the lagging strand would therefore be completely and continuously inhibited across the replication fork.

Predict the immediate outcome on DNA integrity if DNA ligase activity were completely inhibited following the synthesis of Okazaki fragments.

If DNA ligase activity were completely inhibited, the integrity of the newly synthesized DNA would be compromised, particularly on the lagging strand. After the RNA primers are removed and replaced with DNA nucleotides by DNA Polymerase I, DNA ligase is normally responsible for joining the resulting Okazaki fragments together by re-establishing the phosphodiester bonds in the sugar-phosphate backbone. Without this enzyme, the lagging strand would remain fragmented, full of nicks and breaks, leading to an incomplete chromosome copy.

Describe the original central dogma of molecular biology relating DNA, RNA, and protein synthesis.

The original central dogma of molecular biology, proposed by Francis Crick, describes the fundamental and unidirectional flow of genetic information within a cell. This states that the information stored in the DNA sequence is copied into an intermediary molecule, messenger RNA (mRNA), through transcription, and the mRNA then directs the assembly of a specific sequence of amino acids to synthesize a protein, through translation.

If a bacterial cell is prevented from performing transcription, predict the downstream effects on both messenger RNA and protein levels within the cell.

Transcription is the essential process where the genetic information encoded in DNA is converted into messenger RNA (mRNA). If a bacterial cell were prevented from performing transcription, the immediate effect would be the cessation of all new mRNA production, since mRNA is synthesized directly from the DNA template. The downstream consequence would be a rapid decline in new protein synthesis, because the production of all new proteins relies on mRNA carrying the genetic message from the DNA to the ribosomes for translation.

Hypothesize why an RNA virus, such as the influenza virus, must carry its own specific RNA-dependent RNA polymerase in its capsid, rather than relying solely on host machinery.

An RNA virus, such as the influenza virus, must carry its own RNA-dependent RNA polymerase because the host cell's enzymes follow the central dogma and lack the necessary machinery to synthesize a new RNA strand using an RNA template. Specifically, influenza is a negative (-) sense ssRNA virus, and its genome cannot function directly as messenger RNA (mRNA). Therefore, it must use its virally encoded RNA polymerase carried within its capsid to convert the genomic (-) sense RNA into the complementary (+) sense strand, which can then be used as mRNA by the host ribosomes.