MIC 205: 09/16 LECTURE (EXAM 1)

Exam logistics and Thursday material

• Bring identification and required documents: ID, passport, library card (these materials are relevant for the exam information provided).
• Exam format: the entire period will cover material; approximately 45–55 multiple-choice questions.
• Thursday materials: expect structured pictures of reactions and possibly visual aids to accompany the topics.
• Any questions about prior material or Thursday’s content can be asked during class.

Recap: Cellular respiration overview

• Cellular respiration includes glycolysis, pyruvate oxidation, the TCA cycle, and the electron transport chain (ETC).
• Glycolysis:
  • Produces a little ATP and NADH; NADH feeds into the ETC.
  • Pyruvate is formed at the end of glycolysis.
• Pyruvate oxidation:
  • Pyruvate is transformed into acetyl-CoA, producing NADH in the process.
• TCA cycle:
  • Extracts energy from glucose-derived carbon skeletons.
  • Produces NADH, FADH2, and GTP (which can be converted to ATP).
• Overall perspective on the TCA cycle: two views
  • Growth story: carbon skeletons are produced for amino acids, nucleotides, etc.
  • Energy story: flux through NADH/FADH2 to the ETC for ATP production.
• Electron transport chain culminates in oxidative phosphorylation, with a typical ATP yield around ~38 ATP per glucose in aerobic respiration (organism-dependent).

Oxidative phosphorylation and the proton gradient

• Electron carriers (e.g., NADH) donate high-potential electrons to the ETC.
• Proton pumping across the membrane creates a proton motive force or electrochemical gradient.
• Analogy: a car battery with jumper cables creating a potential difference; protons flow back through ATP synthase to drive ATP formation.
• ATP synthase converts the proton motive force into ATP:
  • $ext{ATP yield per glucose in aerobic respiration} \approx 38 \,\text{ATP}.$
• Terminal electron acceptor in aerobic respiration is molecular oxygen, forming water:
  • At the end of the chain, electrons still have a small amount of energy; oxygen accepts them and becomes reduced to water.
• Redox energy scales (conceptual): energy differences drive proton pumping and ATP production.

Redox potentials and electron shuttles

• NAD+/NADH functions as an electron shuttle, carrying high-potential electrons into the ETC.
• Redox potential references from the lecture:
  • Oxygen reduction potential: $E^ ext{°}( ext{O}<em>2/ ext{H}</em>2 ext{O}) = +820\ ext{mV}.$
  • Nitrate reduction to nitrite (anaerobic respiration): $E^ ext{°}( ext{NO}<em>3^-/ ext{NO}</em>2^-) = +421\ ext{mV}.$
  • NAD^+/NADH couple: $E^ ext{°}( ext{NAD}^+/\text{NADH}) = -320\ ext{mV}.$
  • Pyruvate to lactate (fermentation/regeneration): ≈ $E^ ext{°} \approx -180\ ext{mV}.$
• These potentials underlie why different terminal electron acceptors yield different ATP totals (more favorable gradients yield more pumping and ATP).

Anaerobic respiration vs aerobic respiration

• In anaerobic respiration, glycolysis and pyruvate oxidation occur, but the ETC ends with an electron acceptor other than O₂ (e.g., nitrate to nitrite).
• Energy yield differences:
  • Aerobic respiration generally yields more ATP due to a larger potential energy difference to O₂.
  • Anaerobic respiration yields less ATP per glucose than aerobic respiration; commonly discussed values include ~28 ATP per glucose, though numbers can vary by organism and conditions. In some contexts, figures like ~20 ATP are cited; the lecturer notes both, indicating organism-dependent variation.
• Why less ATP with anaerobic respiration:
  • Terminal electron acceptor (e.g., NO₃⁻) has a lower redox potential than O₂, reducing the proton pumping and the proton motive force.
• Summary: aerobic respiration → higher ATP yield; anaerobic respiration → lower ATP yield with alternate terminal electron acceptors.

Fermentation: recycling NAD⁺ and limited ATP production

• Fermentation uses glycolysis as the starting point, but regnerates NAD⁺ so glycolysis can continue when NAD⁺ is scarce.
• Key concept: fermentation regenerates NAD⁺ by reducing pyruvate (or a derivative) using NADH.
• Two main fermentation pathways:
  • Lactic acid fermentation: NADH reduces pyruvate to lactate, regenerating NAD⁺. Occurs in muscle cells and some bacteria.
  • Ethanol fermentation (Saccharomyces cerevisiae and others): Pyruvate is decarboxylated to acetaldehyde, which is then reduced by NADH to ethanol, regenerating NAD⁺. CO₂ is released in this step; explains bread rising due to CO₂ production.
• Why fermentation can be important: allows ATP production via glycolysis when no external electron acceptor is available, but yields only ~2 ATP per glucose.
• Other fermentation products exist (species-dependent):
  • Propionic acid production (Propionibacterium) contributes to Swiss cheese flavor and holes due to CO₂ production.
  • Some Clostridium species produce acetone as a fermentation end product; there are medical/nutritional implications.
• Note on bread rising: CO₂ production during fermentation causes dough to rise; ethanol produced is largely evaporated during baking.

Central metabolic pathways and carbon skeletons

• Central metabolic pathways include glycolysis, pyruvate oxidation, TCA cycle, and the pentose phosphate pathway (PPP).
• Pentose phosphate pathway (PPP) functions:
  • Generates NADPH for biosynthetic reactions and maintenance of redox balance.
  • Produces carbon skeletons such as ribose-5-phosphate (nucleotide synthesis) and erythrose-4-phosphate (aromatic amino acid biosynthesis).
  • Key intermediate sugars mentioned: sedoheptulose-7-phosphate, erythrose-4-phosphate, ribose-5-phosphate, glyceraldehyde-3-phosphate (G3P) and other pentose phosphates.
• PPP serves as a source of carbon skeletons for building nucleotides, amino acids, and glycan precursors, in addition to providing NADPH.
• The central pathways act as hubs where intermediates can be siphoned off to synthesize macromolecules (DNA/RNA, proteins, polysaccharides, lipids).

Catabolic energy sources beyond glucose

• If no glucose is present, cells can extract energy from other macromolecules:
  • Lipids: taken up and broken down; glycerol enters glycolysis as glycerol-3-phosphate, fatty acids undergo β-oxidation to acetyl-CoA; acetyl-CoA enters the TCA cycle.
  • Resulting ATP yield depends on how far the acetyl-CoA enters the pathway; higher entry points yield more ATP.
  • Proteins: imported and degraded into amino acids; some amino acids feed into glycolysis, some into acetyl-CoA, and others into various points of the TCA cycle; ATP yield varies by amino acid.
• The key takeaway: alternative substrates yield ATP, but generally not as efficiently as optimal glucose oxidation.

Anabolism: gluconeogenesis and the Calvin cycle

• When no carbohydrates are available, cells may synthesize glucose de novo via gluconeogenesis (reverse glycolysis) or via the Calvin cycle (CO₂ fixation).
• Gluconeogenesis basics:
  • Starts with pyruvate and builds up to glucose through intermediates such as oxaloacetate and phosphoenolpyruvate (PEP).
  • It requires input of energy (ATP and NADH) because it is effectively running glycolysis in reverse with bypassed steps that require energy input.
  • This process provides carbon skeletons for building nucleotides, glycan sugars, and other cellular components when external carbohydrates are scarce.
• Calvin Benson cycle (carbon fixation):
  • The process by which CO₂ is fixed into organic sugars, akin to running cellular respiration in reverse.
  • It is energy-intensive, requiring 30+ ATP equivalents per turn to build glucose, and typically uses NADPH as reducing power.
  • This cycle is more typical of photosynthetic organisms; in heterotrophs, Calvin cycle-like carbon fixation is less common, but conceptually important for understanding carbon reallocation and autotrophic metabolism.
• Practical note: in carbohydrate-poor environments, gluconeogenesis and, where applicable, Calvin cycle-like processes enable the synthesis of glucose and other carbohydrates needed for cell wall components, nucleotides, and glycan structures; these pathways require substantial energy input.

Lipid, amino acid, and nucleotide biosynthesis from central metabolism

• Lipid biosynthesis:
  • Glycerol-3-phosphate derived from glycolysis forms the glycerol backbone.
  • Acetyl-CoA is the building block for fatty acid tails; these combine to form phospholipids or triglycerides depending on the cellular needs.
• Amino acid biosynthesis:
  • Carbon skeletons are built via central pathways and then amino groups are added to form amino acids.
  • Humans synthesize only a subset of amino acids; the discussion notes that humans can synthesize around 10–12 nonessential amino acids, whereas bacteria often synthesize all 20 amino acids.
• Nucleotide biosynthesis:
  • Requires sugars (e.g., ribose from PPP) and nitrogenous bases (purines and pyrimidines).
  • Central metabolic pathways provide the precursor molecules and energy required to assemble nucleotides.

Regulation of metabolism in microbes

• Core idea: cells regulate metabolic pathways to avoid wasting energy on unneeded processes.
• Preference for energy efficiency:
  • Microbes preferentially use the most energetically efficient carbon source first; glucose is typically preferred.
  • If glucose is present, cells will utilize it first; alternative carbohydrates like lactose are used only after glucose is depleted.
• Regulatory example (lactose utilization in E. coli):
  • When glucose is consumed first, lactose metabolism enzymes are not expressed initially to avoid unnecessary energy expenditure.
  • Upon glucose depletion, the cells upregulate genes for lactose transport and metabolism, including:
  • Lactose transporter (to import lactose).
  • Lactose hydrolase (beta-galactosidase) to cleave lactose into glucose and galactose.
  • Galactose conversion pathway to convert galactose to glucose.
  • This upregulation allows growth on lactose once glucose is exhausted.
• Concept of transcriptional and translational regulation to meet environmental conditions; the cytoplasm remains uncluttered by unnecessary enzymes.

Growth, colonies, and biofilms

• Growth patterns in bacteria:
  • In lab cultures, colonies grow as discrete colonies on plates; derived from single cells through binary fission.
  • Sessile growth: colonies fixed to a surface.
  • Planktonic growth: cells are suspended in liquid; turbidity indicates cell density (e.g., millions of cells per mL).
• Biofilms in nature:
  • Multicellular communities of microorganisms including bacteria, algae, fungi, and protozoa formed on surfaces (e.g., river rocks, dental plaque).
• Doubling time and growth rate:
  • Doubling time varies by species. For example, E. coli can divide roughly every 20 minutes under optimal conditions.
  • Some organisms (e.g., Thiobacillus) can take 1–2 days to divide under less favorable conditions.
• Exponential (logarithmic) growth vs human reproductive patterns.
• Clinical relevance: nosocomial (hospital-acquired) infections often arise in settings with poor aseptic technique or improper lab practices; emphasizes the importance of careful behavior and aseptic technique in healthcare and lab environments.

Aseptic technique, safety, and antibiotic stewardship

• Lab safety and behavior:
  • Most accidents in microbiology labs have been attributed to student behavior and failure to follow safety protocols.
  • Strong emphasis on aseptic technique to prevent contamination and exposure.
• Nosocomial infections:
  • In healthcare settings, preventing infection requires proper sample handling and aseptic procedures to reduce transmission of potential pathogens.
• Antibiotic stewardship and specimen collection:
  • There is a push to identify appropriate specimens before administering antibiotics to combat antibiotic resistance.
  • A shift from empiric antibiotic use to targeted therapy based on proper identification of pathogens.
  • The discussion includes a historical note: fifteen years ago, a large majority of patients were treated empirically, but today there is greater emphasis on appropriate specimen collection and targeted therapy to reduce resistance.

Final connections and Thursday prep

• Central theme: glycolysis, TCA cycle, PPP, and ETC form an integrated network that supplies ATP, reducing equivalents, and biosynthetic precursors for macromolecules.
• The same central pathways feed into both energy production and the synthesis of carbon skeletons for nucleotides, amino acids, and carbohydrates.
• Thursday’s session will likely feature structured visual aids (pictures) of reactions to reinforce these concepts.
• Quick conceptual recap:
  • Aerobic respiration yields the most ATP via ETC and oxidative phosphorylation; terminal electron acceptor is O₂.
  • Anaerobic respiration uses other acceptors (e.g., nitrate), yielding less ATP due to a smaller proton gradient.
  • Fermentation regenerates NAD⁺ to sustain glycolysis when no external electron acceptor is available, yielding only ~2 ATP per glucose.
  • Microbes adjust metabolism based on substrate availability and regulatory networks to optimize energy use (glycolysis first with glucose, then other sugars).
  • Anabolism (gluconeogenesis and Calvin cycle) builds carbohydrates when they are not available, at a high energy cost.

Quick reference: key numbers and concepts

• Approximate ATP yields:
  • Aerobic respiration: $ext{ATP}_{ ext{aerobic}} \approx 38\ ext{ATP per glucose}.$
  • Anaerobic respiration: typically less, around $28\ ext{ATP per glucose}$ (organism-dependent).
  • Fermentation: $2\ ext{ATP per glucose}.$
• Redox potentials mentioned:
  • $E^ ext{°}( ext{O}<em>2/ ext{H}</em>2 ext{O}) = +820\text{ mV}.$
  • $E^ ext{°}( ext{NO}<em>3^-/ ext{NO}</em>2^-) = +421\text{ mV}.$
  • $E^ ext{°}( ext{NAD}^+/\text{NADH}) = -320\text{ mV}.$
  • $E^ ext{°} \approx -180\text{ mV}$ for pyruvate reduction to lactate.
• Carbon skeletons and PPP intermediates:
  • Sedoheptulose-7-phosphate, erythrose-4-phosphate, ribose-5-phosphate (RR, RN, and other PPP products) contribute to nucleotide and amino acid biosynthesis.
• Growth concepts:
  • Doubling time examples: E. coli ~20 minutes; Thiobacter ~1–2 days.
  • Planktonic (free-swimming) vs sessile (biofilm-associated) growth in natural vs lab settings.
• Biological relevance:
  • Biofilms are complex communities contributing to persistence in natural environments and contributing to issues like dental plaque.
• Environmental strategy:
  • When glucose is absent, organisms may use gluconeogenesis or the Calvin cycle to produce glucose, albeit at a high energy cost; in many environments, organisms instead utilize available alternative carbon sources or remain in a state of metabolic regulation until conditions improve.