microm 301: exam 1
Lecture 1
Introduction, Humans and the Microbial World; Chemistry
Independent Study Guide
I. A Little Bit of History
Contributions to Disproving Spontaneous Generation:
Redi, Needham, and Spallanzani
Louis Pasteur, John Tyndall
II. The Scientific Method (Section 1.1)
Hypothesis vs. Scientific Theory
III. Members of the Microbial World (Section 1.3, Figure 1.6)
Organisms vs. Acellular Infectious Agents
Prokaryotes vs. Eukaryotes
Domains: Bacteria, Archaea, Eukarya
IV. Classification/Nomenclature of Organisms (Section 10.1)
Classification – The process of arranging organisms into similar or related groups to provide easy identification and study.
Taxonomic Hierarchies (You only need to know domain, genus, and species):
Species - A group of related isolates or strains
Genus - A collection of related species
Family - A collection of similar genera
Order - A collection of similar families
Class - A collection of similar orders
Phylum/Division - A collection of similar classes
Kingdom - A collection of similar phyla or divisions
Domain - A collection of similar kingdoms
Nomenclature/Scientific Names (Section 1.3)
Genus (first letter capitalized) - Species (lower case)
Italicize or underline the entire name
Strain is sometimes indicated
V. Additional Reading (Section 2.4)
The Molecules of Life (Table 2.4):
Carbohydrates (monosaccharides, disaccharides, polysaccharides)
Lipids (simple lipids, compound lipids)
Proteins
Nucleic Acids (DNA, RNA)
Independent Study Questions:
How did Pasteur’s experiments with the swan-necked flasks contribute to disproving spontaneous generation?
What is the fundamental difference between eukaryotic and prokaryotic cells?
List and describe the two domains of prokaryotic organisms.
In the name Bacillus anthracis, which part indicates the genus and which the species?
Describe the importance and basic structure of proteins, carbohydrates, nucleic acids, and lipids. Provide examples of where they can be found.
Lecture Outline
I. Why Microbiology? (Section 1.2)
Vital Activities of Microorganisms
Essential to other forms of life
The Human Microbiome; Normal Microbiota
Role in the environment
Commercial Benefits of Microorganisms
Food production
Biodegradation
Commercially valuable products from bacteria
Biotechnology
Microorganisms as Model Organisms/Research Tools
Easy to study; findings applicable to other organisms, including humans
Medical Microbiology
Microbes as causes of morbidity and mortality
Understanding their role in disease prevention
II. Living Members of the Microbial World (Section 1.3, Table 1.1)
Composed of cells; generally replicate independently
Bacteria
Prokaryotes
Primary focus of this course; most are beneficial or at least not harmful
Archaea
Prokaryotes; appearance similar to bacteria
Many are extremophiles
Eukarya
Eukaryotes (Table 1.3):
Fungi – Yeasts, molds, mushrooms
Algae – Photosynthetic eukaryotes living in aqueous environments
Protozoa – Single-celled eukaryotes that are not algae or fungi
Multicellular Parasites (Worms) – Live at the expense of a host
III. Non-Living Members of the Microbial World (Section 1.3, Table 1.4)
Acellular; require host cells for replication
Viruses
Nucleic acid (DNA or RNA) surrounded by a protein coat
Viroids
Nucleic acid only (RNA)
Prions
Protein only (e.g., agent causing “mad cow” disease)
Review/Discussion Questions:
Describe the general reasons why microbiology is important.
List and describe the four groups of eukaryotic microorganisms.
Which is more closely related—organisms in the same genus or in the same species?
List and describe the three types of non-living infectious agents.
What are prions?
Lectures 2 and 3
Microscopy and Cell Structure/Function
Independent Study Guide: Day 1
I. Morphology of Prokaryotic Cells (SECTION 1.3)
Shapes (FIGURE 1.8)
Coccus
Rod (Bacillus)
Coccobacillus
Vibrio
Spirillum
Spirochete
Groupings/Arrangements (FIGURE 1.9)
Chains
Packets
Clusters
Multicellular associations
Biofilm containing mixed species (FIGURES 4.2 AND 4.3)
II. Microscopy (SECTION 3.8)
Types of Microscopes
Light Microscopes - maximum magnification ~1000X
Electron Microscopes - maximum magnification ~100,000X
Principles of Light Microscopy
Magnification
Resolution
Contrast
Certain light microscopes can increase contrast (e.g., dark field microscopes, phase contrast microscopes, etc.)
III. Microscopic Techniques: Dyes and Staining (SECTION 3.9; TABLE 3.7)
Wet Mounts vs. Staining
Staining Types
Simple staining
Differential staining
Gram stain (FIGURE 3.14)
Gram-positive – purple
Gram-negative – pink
Special stains (detect specific structures: capsules, endospores, flagella)
Fluorescent dyes and tags
IV. Cytoplasmic Membrane (SECTION 3.1; FIGURE 3.2)
Defines boundary of the cell
Phospholipid bilayer (Fluid Mosaic Model)
Selective permeability (excludes all but water, gases, and small hydrophobic molecules)
Transport Proteins
Control entrance/expulsion of antimicrobial drugs
Function as selective gates
Receptors provide a sensor system
Role in Energy Transformation
Electron transport chain (FIGURE 3.5)
Proton motive force fuels ATP synthesis, flagella rotation, and transport
Independent Study Questions:
What is the advantage of an electron microscope over a light microscope, and vice versa?
What information could one get from a wet mount but not from a simple or Gram stain?
Compare simple, differential, and special stains. Give an example of the last two types.
Describe the shape and color of a Gram-positive coccus vs. a Gram-negative bacillus after Gram staining.
Describe the structure of the cytoplasmic membrane. Where is a cell's cytoplasmic membrane relative to the cell wall?
What is the fluid mosaic model?
List two roles of proteins found in the cytoplasmic membrane.
How is the electron transport chain related to proton motive force?
Lecture Outline: Day 1
I. Transport of Small Molecules Across the Cytoplasmic Membrane
Transport Proteins (FIGURE 3.6)
Transporters, permeases, carriers
Types of Transport Systems (FIGURE 3.7; TABLE 3.1)
Facilitated diffusion – no energy expended
Active transport – energy expended
Some use proton motive force; others use ATP
Group translocation – chemically modifies a compound during transport
Protein Secretion – moves macromolecules outside the cell (FIGURE 3.8)
II. Bacterial Cell Wall (SECTION 3.2; TABLE 3.2)
Function: Provides rigidity, preventing cell bursting
Peptidoglycan: Unique to bacteria (FIGURE 3.9)
Alternating subunits of NAG and NAM form a glycan chain
Glycan chains connected via peptide chains on NAM molecules
Antimicrobial Targets:
Penicillin: Inhibits synthesis
Lysozyme: Breaks down peptidoglycan
Gram-Positive vs. Gram-Negative Cell Wall (FIGURES 3.10, 3.12)
Gram-Positive: Thick peptidoglycan layer, teichoic acids
Gram-Negative: Thin peptidoglycan layer, outer membrane with LPS, periplasm
Mycoplasma species – lack a cell wall
Archaea – variety of cell wall types
Review/Discussion Questions:
What is the role of the cytoplasmic membrane vs. the cell wall?
How is facilitated diffusion different from active transport?
Describe the two general types of active transport systems.
Describe the structure of peptidoglycan.
What are the major differences between Gram-positive and Gram-negative bacteria?
What effect does penicillin have on peptidoglycan?
What effect does lysozyme have on peptidoglycan?
Where is LPS found? What is its medical significance?
Independent Study Guide: Day 2
I. Other Prokaryotic Structures
Capsules & Slime Layers (Glycocalyx Layer)
Function in attachment; pathogenicity (FIGURE 3.12)
Pili (Pilus)
Common pili (fimbriae) – attachment
Sex pilus – involved in DNA transfer
Internal Components
Chromosome vs. Plasmids
Ribosomes – 70S (30S + 50S), target for selective toxicity
Cytoskeleton, storage granules, protein-based compartments
II. Eukaryotic Cell Structure (TABLE 3.4, SECTION 3.7)
Membrane-bound Organelles
Mitochondria & Chloroplasts
DNA sequence similar to Rickettsia and cyanobacteria
Contain 70S ribosomes
Endosymbiotic theory
Independent Study Questions:
What is the difference between a capsule and a slime layer?
What bacterial structures are responsible for motility?
What are fimbriae and sex pili used for?
What is the function of a ribosome? What is the significance of 16S rRNA?
How do eukaryotic structures differ from prokaryotic counterparts (e.g., flagella, plasma membrane, ribosomes)?
What is the medical significance of ribosomal differences?
What is the endosymbiotic theory?
Review/Discussion Questions:
How are capsules related to dental caries?
Describe the general mechanism of chemotaxis.
Describe some characteristics commonly encoded on plasmids.
Name two genera that produce endospores.
Define sporulation and germination in bacterial life cycles.
Compare pinocytosis and phagocytosis.
What is the role of actin in eukaryotic cells?
How have some pathogens hijacked actin machinery?
MICROM 301: Lecture 4
Dynamics of Microbial Growth
Independent Study Guide
I. Principles of Microbial Growth (Section 4.1)
Microbial growth: increase in the number of cells in a population (NOT in size)
Binary fission (Figure 4.1): bacteria and archaea (prokaryotes) multiply; cell length increases then → divides
Exponential growth (Table 4.1): cells multiplying in number exponentially → has important health consequences
When calculating # of bacterial cells over time, consider…
# of cells in original population
# of times cells will divide during stated period
Generation time: time it takes for a population to double in number
Varies between species; influenced by the conditions in which cells are grown
Cell increases in length
DNA moves into each future daughter cell → cross wall forms
Cell divides into two daughter cells → cells separate
II. Environmental Factors That Influence Microbial Growth (Section 4.4)
(Table 4.3 – Know general environments, not exact parameters)
Temperature (Figure 4.8)
Psychrophile: “cold”; optimum between -5*-15*
Grow in arctic/antarctic regions
Psychrotroph: “nourishment”; optimum between 15*-30*
Grow at lower temps; cause spoilage in refrigerated foods
Mesophile: “middle”; optimum between 25*-45*
Grow in soil, colder temps
E.g., e coli, common bacteria, adapted pathogens @ 35*-40*, etc.
Include pathogens (optimum includes human body temp!)
Thermophile: “heat”; optimum between 45*-70*<
Grow in hot springs and compost heaps
Hyperthermophile: “excessive”; optimum of 70*<
I.e., archaea (prokaryotes)
O₂ Availability: O2 Requirements of PROKARYOTES (Table 4.2 – Review definitions)
Obligate aerobe: absolute O2 requirement
Used in aerobic respiration (energy harvesting)
E.g., Micrococcus luteus
Facultative (flexible) anaerobe: grow better in O2, but not required
Use O2 when available, use other types of metabolism when not
O2 respiration is fastest because → it produces the most ATP
E.g., E. coli
Obligate anaerobe: cannot multiply if O2 is present (can be killed by air exposure)
Uses other energy harvesting processes
E.g., most large intestine bacteria
Microaerophile: requires small O2 amounts (2-10%)
Higher O2 contents → inhibitory
E.g., Helicobacter pylori
Aerotolerant anaerobe: indifferent to O2
Can grow in O2, do not use O2 for energy harvesting
Also called obligate fermenters: fermentation is only metabolic option
E.g., Streptococcus pyogenes
pH and Water Availability
Helicobacter pylori is a neutrophile (contrast with acidophile, alkalophile)
Grows in the stomach, decreases acidity of surroundings by producing urease
Urease: enzyme that splits urea into → CO2 + ammonia (neutralizes stomach acid)
Neutrophile: live in and multiply in pH 5-8 (acidic-basic); pH 7 optimum
Most microbes
Cannot withstand highly acidic conditions (with the exception of Helicobacter pylori)
Acidophiles: pH 5.5> optimum
E.g., Picrophilus oshimae (archaea) → pH 1>
Alkaliphiles: pH 8.5< optimum
Live in alkaline lakes/soils
Plasmolysis: Occurs due to water loss in high-salt/sugar environments
Occurs when solute concentration is → medium > cell
Causes osmosis: water diffuses out of cell
→ plasmolysis: cytoplasm dehydrates/shrinks
Used in Food preservation: Use of high salt/sugar inhibits microbial growth
Halophile vs. Halotolerant organisms
Halophile: require high sodium chloride levels
E.g., marine bacteria, archaea, etc.
Halotolerant: tolerate high salt concentrations (up to 10% NaCl)
E.g., Staphylococcus
III. Microbial Growth in Laboratory Conditions (Section 4.3)
Aseptic Technique: Prevents contamination
Sterilization Methods:
Autoclaving – Pressurized steam (Figure 5.4)
Filtration – Removes microbes; used for heat-sensitive solutions (Figure 5.6)
The Growth Curve (Figure 4.6):
Lag Phase – Synthesis of cell components; preparation for division
When transferred to a different medium, cells do not immediately divide; no increase in cell number
Cells begin synthesizing enzymes for growth
E.g., medium with fewer nutrients → longer lag phase
Log (exponential) Phase – Rapid, constant rate of division
Generation time used as a measure
Actively multiplying cells → bacteria most sensitive to antimicrobial meds
Primary metabolites: small molecules made by cells as they multiply
Secondary metabolites: microbial compounds (begin accumulating); purpose other than growth
Stationary Phase – Division rate = death rate
Nutrients levels too low to sustain growth
# of viable cells (capable of developing/multiplying) → constant
Dead cells burst → releases nutrients → fuels the growth of other cells
Death Phase – Constant rate of cell death
Cell death (like growth) is exponential
Phase of Prolonged Decline – Some cells survive and adapt
Cells adapt/tolerate poor conditions → can adapt for a short period (with nutrients from dead cells) → eventual death
Continuous Culture/growth – Important in industrial microbiology
Chemostat: an open system; continuously drips fresh medium → broth culture
Open system: nutrients can be added; waste products can be removed
X volume of fresh medium enters → X volume (cells, wastes, old medium) leaves
Used in industrial processes → harvest commercially valuable products during log phase
Independent Study Questions
If you start with 100 cells and a generation time of 20 minutes, how many cells after 2 hours? How many generations?
Classify microbes based on temperature:
Bacteria on your bench
Bacteria from the Arctic Circle
Bacteria from hot springs
Difference between aerotolerant and facultative anaerobe?
Why are high salt/sugar solutions used to preserve food?
Importance of aseptic technique in microbiology?
Two ways to sterilize media?
Phases of microbial growth? What happens in each?
In which phase is generation time measured?
Lecture Outline
I. Microbial Growth in Nature (Section 4.2)
Biofilms: Communities encased in EPS (extracellular polymeric substances)
Planktonic (free-floating) cells → move to surface & adhere
Bacteria multiply → produce EPS
Extracellular polymeric substances (EPS): accumulation of hydrophilic polymers → creates slippery/slimy surfaces
Other bacteria attach to EPS → grow!
Cells communicate and create architecture
Biofilms have architecture
Channels for nutrients & wastes to pass
Cells communicate through synthesizing chemical signals
Majority of bacterial infections involve biofilms
Also cause industrial maintenance and damages
Development of biofilms (Figure 4.3)
Mixed microbial communities
Microbes in a community compete for nutrients
Can synthesize toxic compounds to inhibit competitors
E.g., contact-dependent growth inhibition: gram-negative bacteria used needle-like structures to inject toxic compounds → competitors
Importance: Environmental, medical, industrial relevance
PRO → Environmental: wastewater treatment, bioremediation, etc.
CON → Medical: dental (plague), contact lenses/cases, etc.
CON → Industrial: piping, tire caps, etc.
II. Environmental Factors That Influence Microbial Growth (Section 4.4)
Temperature, pH, Water Availability (Covered in Independent Study)
O₂ Availability (Tables 4.2 & 4.3)
Obligate aerobe: Requires O₂
Facultative anaerobe: Grows best with O₂ but can grow without
Obligate anaerobe: Cannot grow with O₂
Microaerophile: Requires small O₂; inhibited by higher amounts
Aerotolerant anaerobe: Indifferent to O₂
NOTE: final column → protective mechanisms for each O2 requiremnet
ROS (Reactive Oxygen Species): harmful byproducts of → O2 used in aerobic respiration (metabolism)
E.g., superoxide (O2-), hydrogen peroxide (H2O2), etc.
Cells that grow aerobically must have protection mechanisms
SOLUTION: Detoxified by superoxide dismutase, catalase
Superoxide dismutase: enzyme that inactivates superoxide; converts it to → O2 + hydrogen peroxide
Catalase: enzyme that converts hydrogen peroxide to → O2 + water
Exception: aerotolerant anaerobes
III. Nutritional Factors That Influence Microbial Growth (Section 4.5)
Organisms require: an 1) energy source, and 2) carbon source (+ other required elements)
Energy Source
Phototrophs: obtain energy from sunlight
E.g., plants, algae, photosynthetic bacteria, etc.
Photoautotrophs: obtain energy from sunlight
Photoheterotrophs: obtain energy from sunlight
Chemotroph: extract energy from chemical compounds
E.g., fungi, prokaryotes, etc.
Chemoorganoheterotroph: obtain energy from organic compounds
Chemolithotroph: obtain energy from inorganic compounds
Carbon Source
Carbon fixation: convert inorg C → org C; cycles C in the environment
CO₂: Autotroph: carbon fixation
Photoautotrophs: makes org compounds from CO2
Chemolithotroph: obtain carbon from CO2
Organic compounds: Heterotroph
Photoheterotroph: obtain carbon from org compounds
Chemoorganoheterotroph: obtain carbon from org compounds
Other Elements & Growth Factors
Required by fastidious (complicated nutritional requirements) bacteria
Nitrogen Sources
Nitrogen fixation: converts inorg N → org N → other organisms can easily use N this way
Unique to bacteria & archaea (prokaryotes)
E.g., N2 gas (inorg) → ammonia → incorporate into cellular material (as amino acids) for organisms to use
Phosphorus: often a limiting nutrient in the environment (unable to make as many “batches” as other nutrients)
E.g., cyanobacteria → blue, but toxic (caused by large amounts of phosphate)
Iron: often sequestered in the body → prevents microbial growth
Limiting nutrients: available at the lowest concentration relative to need
E.g., the ingredient that limits how many batches of a recipe can be made
IV. Microbial Growth in Laboratory Conditions (Section 4.3) & Cultivating Microorganisms (Section 4.6)
A. Obtaining a Pure Culture
Pure culture: Descended from a single cell
Allows the study of a single species
Streak plate technique (Figure 4.5): diluting a bacteria on a medium (usually agar plate) to develop colonies
Culture medium: nutrients + sterile necessary
Agar: Used to solidify medium
Not destroyed by high temps
Liquid: 95C
Solid: 45C
Broth culture: Growth measured by turbidity (Figure 4.22a)
Measured by…
Colony formation on solid media (Figure 4.4): develop on a streak plate after incubation
Turbidity in liquid media
B. Culture Media Categories (Table 4.6)
Complex vs. Chemically Defined
Complex: Nutrient broth
E.g., peptone, beef extract, etc.
Defined (minimal): Glucose-salts medium
E.g., glucose, dipotassium phosphate, etc.
NOTE: would not work well for fastidious organisms, more nutrients (e.g., chocolate agar) required
Special Types of Media
Differential Media: Ingredient added to detect changes due to microbial activity
Example: Blood agar (hemolysis – Figure 4.10)
Alpha hemolysis: change the blood cells
Beta hemolysis: lice the blood cells
Ferments: lactose
Selective Media: ingredient added to inhibit growth of unwanted microbes
Example: Media with antibiotics
Selective & Differential Media
Example: MacConkey agar (Figure 4.11)
Selective: contains bile salts + dye → selectively inhibits all but GNR
Differential: Contains lactose + pH indicator → differentiates lactose fermenters = reddish-pink colonies
Other Key Elements
Nitrogen (organic, inorganic, N₂)
Phosphorus/Iron often limiting (see Focus on a Case 4.1)
Iron is sequestered in host – limits microbial growth
Review/Discussion Questions
What is a biofilm? Give an example.
What is a pure culture? How is it isolated?
How do aerobic organisms deal with toxic oxygen derivatives?
Describe 4 classifications of microbes based on energy & carbon source.
Cyanobacteria fix CO₂ and N₂. Which nutrient likely limits growth in aquatic environments?
When would you use a defined medium instead of a complex medium?
What makes a medium differential?
Give an example of:
Selective medium
Differential medium
Medium that is both
If you forget to add a pH indicator to MacConkey agar, the medium is no longer differential.
If a colony grows on MacConkey agar, it is likely Gram-negative.
MICROM 301: Lecture 5
Metabolism
Independent Study Guide (Day 1)
I. Overview of Microbial Metabolism (Section 6.1)
Metabolism: all the chemical reactions in a cell; needing cell components/building blocks + energy (!!!)
Catabolism: degrades compounds → harvest energy → release energy → convert it to a usable form (ATP)
Anabolism (biosynthesis): Synthesize subunits of macromolecules (i.e., amino acids, nucleotides, monosaccharides, fatty acids)
Uses ATP for energy
NOTE: ATP made during catabolism → used during anabolism
NOTE: Catabolism degrades compounds → anabolism uses new parts for synthesis
Harvesting Energy:
Review energy acquisition by photosynthetic vs. chemotrophic organisms (Lecture 4)
Energy Source
Phototrophs: obtain energy from sunlight
E.g., plants, algae, photosynthetic bacteria, etc.
Photoautotrophs: obtain energy from sunlight
Photoheterotrophs: obtain energy from sunlight
Chemotroph: extract energy from chemical compounds
E.g., fungi, prokaryotes, etc.
Chemoorganoheterotroph: obtain energy from organic compounds
Chemolithotroph: obtain energy from inorganic compounds
Exergonic vs. endergonic reactions; energy coupling
Exergonic: reactants free energy > products free energy
I.e., Energy is released →
Endergonic: reactants free energy < products free energy
I.e., ← Energy input needed
Activation energy: energy needed to start a reaction
NOTE: exergonic reactions also have an activation energy
Components of Metabolic Pathways (Figure 6.4)
Metabolic pathway: enzymatically catalyzed chemical reactions; convert substrate → product
Metabolic (biochemical) pathways determined by enzymes (encoded by genes)
Each enzyme converts the substrate for the next enzyme/reaction (until final product is reached)
I.e., substrate I→enzyme 1→substrate II→enzyme 2…→end product
They convert an initial substrate through a series of steps into an end product.
They can be linear, branched, or cyclical.
They are carefully regulated.
They are well-organized and structured.
Pathway types….
Linear: Starting compound → Intermediate(s) → End product
Branched: Starting compound → Intermediate(s) 1,2,3… → End product 1,2,3…
Cyclical: Starting compound → Intermediate(s) → End product → Intermediate(s) → Starting compound…
Enzymes (Figure 6.5): protein catalyst that speed up conversion of substrate → product by lowering activation energy
Each reaction catalyzed by a specific enzyme
Enzyme-substrate complex, active site (Figure 6.12)
Cofactors (Figure 6.13)
Assist enzymes (e.g., Mg, Zn, Cu, other trace elements)
Coenzymes: organic cofactors (Table 6.4)
Can be reused, needed only in small quantities
Environmental Factors Influencing Enzyme Activity (Figure 6.14)
Temperature: 10* rise → doubles speed of enzymatic reactions
I.e., bacteria grow more rapidly at higher temps
Only until optimal activity is reached; too high temps → denatures
pH: slightly above 7; low salt concentrations
Role of ATP (Figures 2.31 & 6.6)
Substrate-level phosphorylation: energy released from exergonic reactions → powers phosphate group addition to → ADP
ADP: receives addition of a phosphate group → forms ATP
ADP + phosphate group → ATP = “energy currency”
ATP → ADP - phosphate group = “energy spent”
Oxidative phosphorylation: energy from proton motive force → powers phosphate group addition to → ADP → can create significant amount of ATP
Reaction catalyzed by → ATP synthase
Proton motive force: energy from electrochemical gradient (by electron transport chain)
Photophosphorylation: light/radiant energy → proton motive force energy → powers phosphate group addition to → ADP
Oxidation/Reduction (redox) Reactions (Figure 6.7): involved in transfer of electrons
OIL: Oxidation is loss of electrons; RIG: Reduction is gain of electrons
Hydrogen often moves with electrons
General rules:
Gain oxygen/lose hydrogen = oxidation
Lose oxygen/gain hydrogen = reduction
NOTE: oxidation = dehydrogenase
Electron Carriers (Table 6.1): where electrons are initially transferred when cells remove electrons from an energy source
NAD+ → loses electrons → NADH: carries 2 electrons + 1 proton
oxidation
NADP+ → accepts electrons → NADPH: carries 2 electrons + 1 proton
reduction
FAD → loses electrons → FADH2: carries 2 electrons + 2 protons
oxidation
Precursor Metabolites (Table 6.2)
Precursor metabolites: Intermediates of catabolism also used → in biosynthesis/anabolic pathways
link anabolic + catabolic pathways
subunits of macromolecules (i.e., amino acids, fatty acids, etc.) can be made from
Understand the concept (do not memorize table)
Independent Study Questions:
What is the difference between anabolism and catabolism?
How do cells couple exergonic and endergonic reactions?
What is an enzyme? What factors influence enzymatic activity?
What about the chemical structure of ATP makes its phosphate bonds "high energy"?
How is substrate-level phosphorylation different from oxidative phosphorylation?
Why must oxidation and reduction reactions always be coupled?
Is removal of a hydrogen atom generally an oxidation or a reduction?
What is a precursor metabolite?
Lecture Outline (Day 1)
I. Principles of Metabolism
Review of metabolic pathways
Role of enzymes
Allosteric regulation (Figure 6.15)
Allosteric site vs. active site
Enzyme inhibition (Table 6.5)
Competitive inhibition (e.g. sulfa drugs, Figure 6.16)
Has a similar structure of substrate → competes to bind with enzyme
Specific to bacteria → good for therapeutic/antibiotic
Non-competitive inhibition:
Regulatory molecules (allosteric)
Allosteric inhibitor: changes active site on enzyme → prevents original substrate form binding
Feedback regulation: Bypasses need for intermediate(s) and enzymes; goes straight from enzyme A → end product
Enzyme poisons (e.g. mercury) → irreversible (enzyme cannot function anymore)
Role of ATP (Figures 2.31 & 6.6)
Substrate-level phosphorylation
Oxidative phosphorylation (via PMF and electron transport chain)
Photophosphorylation (uses radiant energy to generate PMF)
Role of Electron Carriers (Reducing Power):
Glucose + 6 O2 → 6 CO2 + 12 H2O
12 pairs of electrons removed from glucose
Used in:
Electron transport chain (create PMF)
Biosynthesis (reduction reactions)
II. Overview of Catabolism
Overall Reaction:
Glucose + O2 (C6H12O6) → CO2 + H2O + Energy
Carbon energy source: glucose
Terminal electron acceptor: O2
Energy
ATP via substrate-level phosphorylation
NADH/FADH2 → Electron Transport Chain → PMF → ATP (oxidative phosphorylation)
NOTE: reducing power
Pathways:
Glycolysis: glucose → pyruvate
Pentose Phosphate Pathway
Transition Step
TCA Cycle
Cellular Respiration
Fermentation
Energy Source & Terminal Electron Acceptor (Figure 6.10)
III. Central Metabolic Pathways (Section 6.3)
Glycolysis (Figure 6.17): oxidation of glucose → pyruvate
Two stages:
Investment/preparatory stage: 1 glucose (6C) → 2 G3-P (3C)
“Lysing glucose into 2 parts
Uses 2 ATPs
Payoff phase: G3P (3C) → pyruvate (3C)
Produces 2 ATPs + 1 NADPH
TOAL: 4 ATPs + 2 NADHs per glucose molecule
NET: 2 ATPs + 2 NADHs
Note: precursor metabolites
Pyruvate undergoes inter step before → entering TCA cycle
TCA cycle: each acetyl coa enters → TCA cycle
Oxidized → 2 CO2
Produces → 3 NADH + 1 FADH2 + 1 ATP
Note: central metabolic pathway that generates the most reducing power (NADH/FADH2) → that will be turned into ATP
Pentose Phosphate Pathway
Glucose → intermediate of glycolysis
NADPH (amount varies)
2 precursor metabolites
Transition Step (Figure 6.18)
Pyruvate (3C) → Acetyl CoA (2C) + CO2
NADH
1 precursor metabolite
TCA Cycle (Krebs, Citric Acid Cycle) (Figure 6.18)
Acetyl CoA → 2 CO2 (x2 per glucose)
ATP
3 NADH
1 FADH2
2 precursor metabolites
Review/Discussion Questions:
What is the mechanism of action of sulfa drugs?
Mercury kills bacteria because it is a non-competitive enzyme inhibitor. What does this mean?
What is the fate of electrons carried by NADH and FADH2? What about NADPH?
What are the central metabolic pathways? Why are they called central?
What is the endpoint of glycolysis? Of the TCA cycle?
What is the transition step?
Why must the transition step and TCA cycle repeat twice per glucose molecule?
Identify which central metabolic pathway: a. Can initiate the breakdown of glucose? b. Is generally used exclusively for biosynthesis? c. Generates the most reducing power? d. Generates the most ATP directly? e. Includes steps that use ATP?
NOTE: ETC (ELECTRON TRANSPORT CHAIN) & REDUCING POWER
MICROM 301: Lecture 6 – Metabolism (Day 2)
Independent Study Guide (Day 2)
I. Review Redox Reactions
Oxidation/Reduction (redox) Reactions (Figure 6.7): involved in transfer of electrons
OIL: Oxidation is loss of electrons; RIG: Reduction is gain of electrons
Hydrogen often moves with electrons
General rules:
Gain oxygen/lose hydrogen = oxidation
Lose oxygen/gain hydrogen = reduction
NOTE: oxidation = dehydrogenase
II. Using Precursor Metabolites in Biosynthesis (Figure 6.28)
Calvin cycle: complex cycle with “6 turns”; produces one molecule of 6-carbon sugar → fructose-6-phosphate (i.e., 18 ATP, 12 NADPH, 6 CO2)
Incorporating CO2 into → org compound
Starts a new round of the cycle
Reducing resulting molecule
ATP + NADPH → reduces stage 1 product
→ produces org compound used in biosynthesis
Regenerating starting compound
Org compound is regenerated to continue cycle
No need to memorize which exact precursor metabolite makes which specific biomolecule.
Just know which pathway contributes to their synthesis (NOTE: PG 152)
Example: LPS comes from a precursor metabolite made in glycolysis.
Glycolysis → precursor metabolite → ___
LPS
Peptidoglycan
Lipids (glycerol comp)
Proteins (amino acids: cys, glyc, ser)
Proteins (amino acids: phenyl, trypto, tyro)
Also: pentose phosphate cycle
Proteins (amino acids: alan, leuc, val)
Pentose phosphate cycle → precursor metabolite → ___
Nucleic acids & proteins (amino acid: histi)
Proteins (amino acids: phenyl, trypto, tyro)
Also: glycolysis
Transition step → precursor metabolite → ___
Lipids (fatty acids)
TCA cycle → precursor metabolite → ___
Proteins (amino acids: arg, gluta, pro)
Proteins (amino acids: aspart, apsara, isoleuc, lys, methio, threo)
III. Catabolism of Organic Compounds Other than Glucose (Section 6.6)
Compounds enter the central metabolic pathways as precursor metabolites (Figure 6.24)
Exoenzymes:
Polysaccharides & disaccharides:
Lipids:
Proteins:
Independent Study Questions (Day 2):
Where do the electrons carried by NADH enter the electron transport chain relative to those carried by FADH2?
Which is the only pathway that provides a precursor metabolite for nucleotide biosynthesis?
What parts of the central metabolic pathway provide precursor metabolites for lipid synthesis?
How would a bacterium use protein as an energy source?
How would a bacterium use lipids as an energy source?
Lecture Outline (Day 2)
I. Cellular Respiration (Section 6.4)
The central metabolic pathways oxidize glucose — but where do the electrons go?
Electron Transport Chains:
Mitochondrial ETC (Part of Figure 6.19)
E. coli ETC (Figure 6.20)
ETC → use of proton motive force
Prokaryotes → a lot of ETC variation
Aerobic respiration: shown in image
Anaerobic respiration: different enzymes, fewer protons ejected → less proton motive force (??)
NO3 as TEA
Note: oxidase test
Respiration Types: ^^ see above
Aerobic Respiration
Anaerobic Respiration
NO₃⁻ is the terminal electron acceptor (TEA)
Energy Yield of Glucose Metabolism (Aerobic Respiration):
4 ATP – substrate-level phosphorylation
10 NADH
2 FADH₂
Theoretical max = ??
Substrate level = 4 ATP
Oxidative = 34 ATP
Energy Source vs. Terminal Electron Acceptor (Figure 6.7):
Glucose + 6 O₂ → 6 CO₂ + 12 H₂O
II. Application: Microbiology of Wastewater Treatment (Section 29.1)
BOD (Biochemical Oxygen Demand):
Amount of O₂ required for microbial decomposition of organic matter in a sample
H2O ~ 5-10mg/liter dissolved in O2
O2 is converted → H2O
Electron Transport Chain → Oxidative Phosphorylation
Municipal Wastewater Treatment Methods:
Primary: physically removes material that settles out
Eliminates ~50% of solids
25% of BOD
Secondary: converts solids → inorg compounds + cell mass that can be removed
Eliminates ~95% of BOD (most pathogens)
Note: benefits of biofilms
Advanced: purification beyond secondary
Removes nitrates and phosphates
III. Fermentation (Section 6.5, Part of Figure 6.10)
Used by cells when respiration is not an option (no TEA or no ETC) or when unable to perform respiration
Produces less energy than respiration
Energy is conserved as chemical energy in end products
Glucose oxidation stops at pyruvate
Electrons from NADH passed to pyruvate or a derivative
End Products of Fermentation (Figure 6.23)
Note: test tube products are being used to “ID” molecules
IV. Review (Table 6.3)
Logic of Fermentation:
Oxidizes NADH to regenerate NAD⁺ for continued glucose breakdown
Skips transition step and TCA cycle (which generate 5x more reducing power)
Review/Discussion Questions:
What is proton motive force used for in the mitochondria? In bacteria?
In the breakdown of glucose, which generates the most ATP: substrate-level phosphorylation or oxidative phosphorylation?
How is fermentation different from respiration?
How is NAD⁺ regenerated in respiration vs. in fermentation?
How is aerobic respiration different from anaerobic respiration?
When glucose serves as the energy source, which terminal electron acceptor—NO₃⁻ or O₂—results in the release of the most energy?
What is BOD? How will adding a substance with a high BOD affect a body of water like Greenlake?
In municipal water treatment systems, what is the difference between primary and secondary treatment? Why is aeration important in secondary treatment?
Give three examples of why the end products of fermentation are important.
MICROM 301: LECTURE 7
Microbial Diversity (Dr. Kendall Gray)
Ubiquity of microorganisms …
Chemical Energy Sources (& Terminal Electron Acceptors)
Chemoorganotrophic metabolism: glucose as an energy source, oxidized to…
Terminal electron acceptors…
Pyruvate: greatest tendency to give up electrons
NO3- → NH4+: moderate tendency to give up electrons
O2: lowest tendency to give up electrons
Chemolithotrophic metabolism: inorganic energy sources, oxidized to…
H2: greatest tendency
H2S: moderate tendency
Fe2+: lowest tendency
Terminal electron acceptors:
H2 → CO2: greatest tendency
H2S & Fe2+ → O2: lowest tendency
Requirements of All Living Organisms
Energy:
Photo: light
Phototrophic: using light for energy
Photosynthetic: using light for energy + biosynthesis (to fix CO2)
Note: lithoautotrophs
Photosystem I: produces oxygen
Photosystem II: produces sulfur
Anoxygenic photosynthesis: no oxygen
Chemo: chemical bond; oxidation of electron donors (i.e., org/inorg compounds)
Electrons (reducing power):
Litho: using inorg compounds for electrons
Note: phototrophs
Organo: using org compounds for electrons
Carbon:
Auto: CO2 carbon source (makes own carbon by fixing CO2)
Reduces CO2 → org compounds
Hetero: Org compound carbon source (unable to make own carbon by fixing)
Oxidizes org compounds → CO2
Types of Bacteria (Microbial Diversity) & Metabolism
Heliobacteria: Type I anoxygenic phototrophs; only gram-pos, phototrophs known
Gram-pos: thick peptido layer, no outer membrane
Energy source
Phototroph: light
Carbon & Electron source
Obligate anaerobes: Lack CO2 fixing → require org compounds for carbon + electron source
do not thrive/grow in the presence of O2
Aka: photoorganoheterotroph
Purple non-sulfur bacteria: Type II anoxygenic phototrophs
Energy source
Phototroph: light, OR
Chemotroph: in the absence of light, respires org compounds
Carbon source
Autotroph: fix CO2, OR
Heterotroph: org compounds
Electrons source
Lithotrophs: sulfide, OR
Organotroph: org compound
Haloarchaea: obligate halophiles (growth requires saturated salt concentrations)
Membrane contains halorhodopsin: contains retinal photopigment → light-driven proton pump
phototrophs… but not photosynthetic
Haloarchaea cause “pink” color of rock salt
Proton motive force from inside → outside of cell
Caulobacter prosthecae: extensions of cell envelope (tipped with adhesive proteins)
Caulobacter life cycle
Swarmer cell:
Stalk initiation:
DNA replication (mother cell):
Cell growth:
Stalked + swarmer cell (daughter cell):
Stalk cells = immobile
Swarmer cell = mbile
Caulobacter cells are morphologically polar, with distinct protein structures at both the "nose" and the "tail".
Swarmer cells have both catabolism and biosynthesis, but do not grow and do not replicate their DNA.
Differentiation into a stalked cell appears to be triggered by an internal "clock" rather than by environmental cues.
The stalk always forms at the "tail" (flagellated) end of the cell. Daughter swarmer cells always elaborate their flagella at the opposite end.
The stalk of the "mother" cell continues to elongate with each round of cell growth and division.
Oligotrophic (low nutrient) conditions: stalk increases surface-volume ratio
→ more efficient uptake of limited nutrients
Holdfast (of rosetta) → as efficient as swimming when accessing limited nutrients (more favorable energetically)
Swarmer progeny cells production → dissemination mechanism → limits competition for scarce resources
Redox reactions
Carbon
Heterotrophs: oxidizes org compounds → CO2
CO2 = most oxidized
Autotrophs: reduces CO2 → org compounds
Org compounds = most reduced
Inorg nutrient cycle: sulfur (respiration)
Sulfate reducers: SO4-2 = electron acceptor (respiration of org compounds)
SO4-2 = most oxidized
Sulfide oxidizers: HS- = electron donor (respiration + CO2 fix)
HS- = most reduced
Inorg nutrient cycle: nitrogen (fixation)
Nitrate reducers: NO3- = electron acceptor (respiration of org compounds)
NO3- = most oxidized
Ammonia oxidizers: NH4+ = electron donor (respiration + CO2 fix)
NH4+ = most reduced
Note: Thiomargarita namibiensis
Able to be sulfur & nitrogen oxidizers (in the mud) and oxygen oxidizers (in the water)
Stores nitrate in their hollow space
Not oxygen because… ??
Bacterial development cycles
Myxobacteria: GNR, obligate anaerobes
Myxo = slime; gliding motility
Degrade complex polymers → growth substrate
Fruiting body: formation of complex, multicellular structure (in response to starvation)
Myxospore: metabolically inert form, differentiated from fruiting body
Cyst: has thousands of myxospores → disseminated as a group
Bdellovibrio bacteriovorus: obligate, intracellular predators
Attack phase: high rates of catabolism (harvesting energy from broken down compounds) & biosynthesis (creating new compounds from broken down parts)
Do not grow & replicate DNA
Invasion: release of exoenzymes → partially degrade prey cell envelope
Penetrates outer membrane/peptido of prey cell
“Bdelloplast” stabilization: release of exoenzymes → covalently modify prey cell envelope
Modification → prevents lysis (splitting) of host cell & block secondary invasion (“this is mine, I claim my territory”)
Growth phase: elongates → aseptate filament (uses cytoplasm of prey cell as substrate)
Differentiation & release: triggered by prey cell depletion (not simple nutrient limitation)
Food Microbiology – Lecture 8 Outline
Beneficial Use of Microbes in Food Productions
Factors influencing microbial growth in food and appropriate storage of food
Microbes in Food and Beverage Production
Yeast – Ethanol/CO₂ – beer, wine, bread
Glucose ferments → pyruvate ferments → ethanol/CO2
Starch = polymer of glucose
Lactic acid bacteria – Lactic acid – cheese, pickles, yogurt, cured meats
Propionibacteria – Propionic acid, acetic acid, CO₂ – cheeses
Focus on Lactic Acid Bacteria
Making of Cheese
Lactose → Pyruvate → Lactic acid
Acid curdles the milk, curds are separated
Lactic acid bacteria
Add flavor, inhibits growth of spoilage org/foodborne pathogens
Obligate fermenters (produce lactic acid)
Lactose → pyruvate → lactic acid
In milk:
Cheese unripened
Cheese: starter cultures → ferment lactose in milk → coagulate proteins
Curd: heated, cut, liquid drained
Cheese ripened (storing for a long time)
Taste changes from metabolic activities of lattice acid bacteria
Long → more acidic/sharper
Aging → slow drying → concentrates flavor
Yogurt
Evaporated milk → becomes concentrated
→ inoculated with → Streptococcus, thermophilus, lactobacillus delbrueckii
Acidophilus milk: fermentation by Lactobacillus acidophilus
Culture added immediately before packaging
Can potentially colonie gut bacteria
Other Fermented Foods
Sauerkraut
Pickles
Poi
Olives
Kimchee
Fermented Meat Products
Add sugar to meat (sugar fermentation) → then dry
Lactic acid bacteria ferments → produces lactic acid → prevents other microbes from growing in the meat
Can smoke/heat to kill other bacteria
Wine Making
Yeast converts sugars → alcohol (fermentation)
Foodborne Illness
Two Major Classes
Foodborne Infection: ingest live microbes → grow in intestinal tract → causes disease
Foodborne Intoxication: toxin produced by microbes → causes disease
Foodborne Intoxication
Botulism: Clostridium botulinum
Endospore forming GPR, obligate anaerobe
Does not grow below pH 4.5 (more likely to grow in non-acidic foods)
Toxin: 1g can kill millions of people
Blocks nerve transission → muscles (flaccid paralysis)
Heat labile: heat it up to kills bacteria/toxins
Staph food poisoning (Staphylococcus aureus)
GPC, tolerates low water availability
Toxin: strain produces toxin causing sickness
heatstable/survives cooking
Some toxins are heat-stable and some are heat-labile
Foodborne Infection
Most frequent types in the U.S.:
Salmonella
poultry
Campylobacter
poultry
E. coli (different serovars with different symptoms and severity)
Comes form meat (e.g., hamburger), manure, etc.
Norovirus
Self-Study Questions
How does adding salt or sugar to food help to preserve it?
How does fermentation with lactic acid bacteria help to preserve foods?
Citrus fruit is acidic. When it spoils, it’s most commonly spoiled by molds.
What can you infer about the growing conditions tolerated by some molds relative to most bacteria?When apples are commercially stored, we often store them in an environment with increased levels of CO₂.
How does this help keep them fresh?
Intrinsic factors influencing microbial growth in food:
Water availability
Can decrease water availability by → adding sugar/salt & drying
Water availability → greater microbial growth
E.g., ham water availability = 0.91 → more likely to grow Staphylococcus
pH
Nutrients
Biological barrier (rinds, skins, etc.)
Antimicrobial chemicals (natural or added)
Extrinsic factors influencing microbial growth in food
Temperature
Refrigeration → slows growth
Freezing → eliminates mot growth
Atmosphere
Reduced oxygen prevents → growth of obligate aerobes
Respiration
Requires O2 → produces CO2
Reaction rate increases → reactant concentration increases
Decreased O@ → increased CO2 → limits microbial growth
Note: glycolysis, TCA cycle, respiration