Bacterial Growth and Molecular Biology Techniques

Central Dogma & Bacterial Replication

• The central dogma describes the flow of genetic information: DNA is transcribed into mRNA, which is then translated into protein/enzyme.
• Bacteria often replicate rapidly within a host, leading to a quick doubling-time to facilitate invasion.

Molecular Assays

• Molecular assays stem from the central dogma and can measure gene expression or enzyme activity.
• Scientists select assays based on the specific biomolecule or process they intend to measure (e.g., DNA replication or gene expression).
• Assays like viability assays (survival), growth curve assays (growth rate), qPCR (mRNA), and enzyme assays provide direct or indirect measurements of the central dogma in response to a manipulated variable.

Bacterial & Viral Interactions

• Beneficial interactions occur when the rate of binary fission exceeds the rate of death.
• Neutral interactions occur when the rate of binary fission equals the rate of cell death.
• Harmful interactions occur when the rate of cell death exceeds the rate of binary fission.

Bacterial Batch Culture Phases

• Lag phase: Bacteria adjust to new culture conditions.
• Log phase: Bacterial population experiences maximum growth and metabolic activity.
  • Ideal for physiological or metabolic studies due to physiological uniformity.
  • Binary fission rate is at its maximum.
• Stationary phase: Population size remains constant due to limited resources.
  • Cell population is physiologically less uniform.
  • Nutrients become limited and metabolic wastes accumulate.
• Death phase: Death rate exceeds binary fission rate because of starvation and harmful levels of metabolic waste.
  • Metabolic waste levels become harmful to cells.
  • Rate of cell death increases.

Stationary Phase & Cell Division

• During the stationary phase, cell division doesn't alter population size.
• Limited nutrients, accumulating metabolic waste, and reduced energy availability slow binary fission. Cell population becomes physiologically less uniform.

Viable Cell Count vs. Turbidity in Death Phase

• Viable cell count measures living cells by counting colonies on a streak plate, where each colony represents a cell.
• Turbidity measures the cloudiness of a liquid culture, reflecting both living and dead cells.

Microbiology Assay Variables

• Manipulated variable: Changed by the researcher (e.g., gene mutation).
• Response variable: Changes in response to the manipulated variable and is measured in the experiment (e.g., CFU on a plate).
• Controlled variable: Maintained constant to accurately measure the impact of the manipulated variable on the response variable (e.g., consistent experimental setup).

Planning a Microbiology Investigation

• The order of planning an investigation: Observations → Question → Hypothesis → Experiment (including control group) → Observations (experimental data supports or doesn’t support hypothesis).

Microbiology Assays

• Viable count method: Involves serial dilutions, plating, incubation, and colony counting.
• Growth curve: Measures optical density (OD) of samples from a fresh culture over time and plots the growth curve.
• Disk diffusion susceptibility test (Kirby Bauer): Plate bacteria, place antibiotic disks on agar, incubate, and measure zones of inhibition.

Bacterial Population Measurement Methods

• Viable Count:
  • Counts only living cells and can distinguish different species, but is time-consuming and may underestimate numbers.
• Growth Curve:
  • Fast and based on real-time measurement, but measures both dead and alive cells, reducing accuracy.

Nutrients

• Macronutrients: Required in large amounts (e.g., Mg, C, H, N, Ca, N, K).
• Micronutrients: Required in small amounts (e.g., Fe, Mn, Co, Cu, Zn, N).
  • Often contaminants in water for growth media.
  • Enzymes use metal ions as cofactors for stabilization and electron transfer.

Sterilization

• Autoclave: Utilizes heat and high pressure (pressure cooker) to kill microorganisms and prevent evaporation.
• Filtration: Used for solutions of chemicals not stable to heat, such as antibiotics.

Culture Media

• Defined medium: All exact chemical species are known and in a known concentration.
• Complex (undefined) medium: Contains ingredients of unknown chemical composition.
• Minimal medium: Contains the minimum number of chemicals needed for growth; all proteins, nucleic acids, lipids, and vitamins must be synthesized. (Slow Growth)
• Rich medium: Contains many preformed building blocks (amino acids, nucleic acids, lipids, etc.) needed for biochemical pathways. (Faster Growth)

Selective vs. Differential Culture Media

• Selective medium: Inhibits the growth of certain microbes while allowing others to grow (e.g., EMB agar for gram-negative bacteria).
• Differential medium: Displays different phenotypes for different microbes, aiding in their identification.

Pure Culture Isolation Methods

• Streak plate: Smears cells on an agar surface to obtain isolated colonies on top of the agar.
• Agar dilution tube: Isolates colonies of anaerobic microbes embedded in the agar.
• Bacterial colonies are considered pure cultures because they originate from a single species of identical cells.

Enrichment Culture

• Enrichment cultures use growth media to enhance the growth of a specific subset of microbes.

Bacteria

• Bacteria are ideal models for determining fundamental life processes due to their many traits that make them good for experimental manipulation.

E. Coli Traits

• E. coli traits have made it an ideal model:
  • Single cellular
  • ~4300 protein coding genes
  • Asexual
  • Minimal living requirements
  • ~20 min generation time
  • Quick, cheap, easy of genetic manipulation
  • Mechanisms like humans (genes and cellular process conserved)

Central Dogma

• DNA transcribed by RNA polymerase into mRNA, then mRNA translated by ribosomes into protein.
• Nucleic acids (DNA & RNA), carbohydrates, proteins, and lipids have conserved functions in eukaryotes and bacteria.

Macromolecule Functions

• Nucleic acid (DNA/RNA): Encodes instructions for synthesis of macromolecules and cell metabolism.
  • DNA stores genetic information for RNA and enzyme synthesis.
  • RNA transfers information from DNA, acting as a template for enzyme synthesis.
• Carbohydrates: Energy and carbon source, backbone of nucleic acids.
• Proteins: Enzymatic activity for energy production and synthesis of macromolecules.
  • Transport nutrients, facilitate enzymatic pathways (catabolic/anabolic), and provide cellular structure and motility.
• Lipids: Fatty acids for energy and carbon storage; phospholipids form membranes.

Genotype vs. Phenotype

• Phenotype: Physical features.
• Genotype: Genetic makeup; determines the phenotype.

Transcription & Translation Similarities

• Key similarities between bacteria and eukaryotes:
  • DNA transcribed into mRNA.
  • mRNA translated into proteins.

Transcription & Translation Differences

• Bacteria: Transcription and translation coupled due to lack of intracellular structures.
  • Nucleoid: Chromosome localized in cytoplasm; site of transcription and translation.
  • Generate ATP without mitochondria.
• Eukaryotes: Transcription and translation separated processes due to intracellular structures.
  • Nucleus: Site of RNA synthesis (transcription).
  • ER: Site of translation (protein synthesis).
  • Mitochondria: Specialized organelle for cellular respiration.

Eukaryote vs. Bacteria

• Shared characteristics:
  • Cell membrane (phospholipid bilayer).
  • Genetic material (DNA).
• Unique eukaryotic structures:
  • Mitochondrion.
  • Organized DNA in chromosomes.
  • Endoplasmic reticulum.
  • Nucleus

Metabolic Pathways

• Aerobic respiration (cellular respiration): 38 ATP.
• Anaerobic respiration: 4 to 36 ATP.
• Fermentation: 4 ATP.

Bacteria

• DNA codes the genotype, transcribed into mRNA, then translated into protein/enzymes, forming the phenotype which drives bacteria metabolic pathways, dictating viable environments.

Cell Morphologies

• Coccus: Single round cell.
• Diplococcus: Two round cells.
• Streptococcus: Chain of round cells.
• Staphylococcus: Cluster of round cells.
• Bacillus: Single rod-shaped cell.
• Spirochete: Spiral-shaped cell.
• Filamentous: Thread-like cell.
• Vibrio: Curved, bean-shaped cell.

Gram Staining Procedure

• Spread culture over slide and air dry.
• Heat fix and flood stain with crystal violet (purple).
• Add iodine as mordant.
• Decolorize with alcohol.
• Counterstain with safranin (pink).
• Gram-positive cells appear purple, and Gram-negative cells appear pink.

Gram (+)

• Gram-positive cells have thick peptidoglycan.
  • 10-15 sheets.
  • Many peptidoglycan crosslinks.
  • Has teichoic acids and lipoteichoic acids

Gram (-)

• Gram-negative cells have thin peptidoglycan.
  • 1-3 sheets.
  • Few peptidoglycan crosslinks.
  • Has lipoprotein

Gram Stain Results

• Differences in cell walls produce gram stain results.
• Gram-positive cells retain crystal violet due to thick peptidoglycan layer.
• Gram-negative cells lose crystal violet during decolorization due to thin peptidoglycan and are then stained pink by safranin.

Peptidoglycan

• Structure: A mesh of polymers of alternating amino sugars connected by peptide crosslinks.
• Role: Forms the murein sacculus unique to bacteria.

Gram Positive vs Gram Negative Cell Walls

• Gram-positive cell wall: Thick peptidoglycan layer with teichoic acids for negative charge and binding positive ions; lipoteichoic acids link to membrane lipids for stability.
• Gram-negative cell wall: Thin peptidoglycan layer with outer membrane containing phospholipids, lipoproteins, and lipopolysaccharide (LPS).

Gram-Negative Structure

• Lipopolysaccharide (LPS): Sugar chain connected to fatty acid chains embedded in the outer membrane.
• Inner face: Phospholipids and lipoproteins.
• Transport proteins: Span bilayer for molecule transport.
• Periplasm: Contains peptidoglycan, enzymes for synthesis of peptidoglycans, and nutrient transport proteins.

Lipopolysaccharide (LPS)

• Lipid A: Disaccharide with fatty acids connected to outer membrane base.
• Core polysaccharide: Connects lipid A to O-specific polysaccharide.
• O-specific polysaccharide: Acts as antigen for identification via stereotyping.

Lipoprotein & Porin Functions

• Lipoprotein: Connects outer membrane to peptidoglycan.
• Porins: Trimeric proteins that span the outer membrane for nutrient transport.

Bacterial Cell & Energy

• Prokaryotes can contain sulfur granules as a source of potential energy.

Nucleoids

• Bacterial cells store DNA in nucleoids through supercoiling by topoisomerases, reducing space.

Spirochete Motility

• Spirochetes move using endoflagella, rotating the entire cell like a corkscrew.

Storage Polymers

• Advantages: Bacteria synthesize storage polymers when food is available and depolymerize them for food when scarce.
• Energy status:
• High energy promotes synthesis, and low energy promotes depolymerization.
• Glycagon: Carbon storage
• Polyphosphate: Phosphate and energy storage.

Gas Vesicles

• Gas vesicles give buoyancy to photosynthetic aquatic bacteria. They can be used to control floating height on water.

Bacterial Spore

• Sporulation occurs in response to bad environmental conditions.
• Advantages: Endospores are resistant to heat and chemicals and can germinate once conditions improve.

Spore Forming Bacteria

• Bacillus and Clostridium are common spore-forming bacteria.
• B. thuringiensis makes biological insecticide; B. subtilis is used in labs to sporulation/germination; B. anthracis causes anthrax.
• C. perfringens causes gas gangrene; C. tetani causes tetanus; C. botulinum causes botulism, all harder to disinfect.

Sporulation Steps

• Vegetative cycle
• Asymmetric cell division to separate cell into mother cell forming prespore
• Septum formation
• Engulfment of prespore by mother cell creating 2 membranes
• Cortex formation
• Spore Coat Formation
• Cell Maturation and Cell Lyse.

Spore Proteins

• Dipicolinic acid: Forms matrix with calcium to stabilize/protect spore
• SASP: Stable to heat, chemicals, and radiation.
• Ca+: stabilise and protect spore

Capsules vs. Slime Layers

• Capsule: Semi-rigid matrix surrounding cell, made of polysaccharides, with a glycocalyx.
• Slime layer: Non-rigid, easily deformed.
• Fimbriae and pili facilitate adhesion, whereas flagella facilitates motion.
• Functions:
  • Physical protection.
  • Protects from desiccation.
  • Protects from immune systems.
  • Adhesion

Slime Layers & Capsules

• Slime Layer: Bacteria that form a slime layer makes a large connection (looks like coral with spots).
• Capsule: Bacteria that form a capsule have a rings around each individual cell.
• Observed with using microscopy and India ink.

External Appendages

• Fimbriae are made of protins that extend outwards from the cell to adhere to host cells and tissues.
• Pili protein tubes that adhere and pass genetic information between bacterial cells. Can extend or shorten.
• Flagella are a helical hallow appendage that rotate to provide motion

Appendages & Host

• Can attach to surfaces which help to form biofilms.
• Host specificity- receptors only found on specific hosts
• Tissue tropism- binding to specific tissue

Rotation

• Directional flagella rotation: unidirectional or bidirectional.

Fimbriae vs. Pili vs. Flagella

• Fimbriae: Small, numerous, adhesion
• Pili: Medium, conjugation, twitching motility
• Flagella: Large, Motility rotation.

Flagella Arrangements

• Polar: One flagella at one end.
• Lophotrichous: Many flagella at one end.
• Peritrichous: Many flagella all around.

PMF, Mot protein, Flagella Rotation

• PMF: Source of energy for rotation.
• Mot proteins: Convert PMF into rotation.
• Rings: What rotates (rotor)

Septum

• In the process of binary fission of a dividing rod-shaped bacterial cell, the septum can be described as the dip between the 2 cells
• FtsZ proteins- the ring of polymerized proteins that form the septum

FtsZ & Min in Cell Division

• The roles of the Min an FtsZ proteins is to help assemble divisome complex in the center of the cell. Assembles DNA and forms the septum to split the cell into membranes
• A ring of 5 FtsZ, depolymerizes to turn GTP into GDP Pi which turns the ring into 4 FtsZ to make it smaller and it keeps going until there are 2 cells

Cell DNA

• Rapidly dividing cells have multiple bidirectional forks so cells can have multiple chromosome copies per cell

MreB

• MreB role is to find non coccoid bacteria Like actin in eukaroytic cell where it forms helica filaments on inner surface to cytoplasmic membrane. Touch points signal points for membrane synthesis

Synthesis

• The synthesis of precursor units of NAG and NAM occur in the cytoplasm during the replication of bacteria
• Enzymes that cut peptidoglycans to create holes in cells and holes are filled by cells adding new precursor units

Bacterial cell Growth

• Bactoprenol allow the cell to move between precursor units and preptidoglycan, and helps the proteins to be transpeptidases
• Autolysins are used by the bacterial cell to cut the peptidoglycans to create holes to fill with precursor while the two are being worked on from by proteins

Penicillin & Bacitracin

• Bacitracin inhibits the Bactoprenol function , this disallows the movement of precursor from inside to outside the cell
• Penicillin inhibits the transpeptidase to the point that percusor and peptidoglycan do not have connecting petides

Metabolic

• DNA replicated gets transcribed to mRNA, then proteins are translated into protein that depend on oxidation and reduction for ATP, DNA< RNA and protein is anabolic, which uses energy from catabolic reactions

Electrons

• Electron donors always on the right, and on the opposite sit are acceptance
• As electrons move, energy increases
• Differences in redox output are based on the the potential eneregy the electrons are moved
• Anaerobic respriation is found only in bacteria

Electrons, Enzymes & Catabolic Metabolsim

• The function of electron carriers and enzymes is to help move catabolic metabolism.
• Iron is used to move the electrons and protons, and the branching is accepted to move these said ions
• ATP powers cells

Glycolysis

• Glycolysis is a catabolic reaction, creating the movement of glucose to pyruvate though reactions that uses electrons to produce more than is used. So 2 is used but 4 are created in the process
• All the electrons produce 2 ATP

Energy Towers & Metabolic Rates

• Fermentation makes 2 ATP from SLP by having glucose as e- donor and pyruvate as e- acceptor
• Aerobic respiration makes 38 ATP from oxidative phosphorylation by having glucose as e- donor and O2 as e- acceptor
• Anaerobic respiration makes from 4 to 36 ATP through oxidative phosphorylation and having glucose as e- donor and NO3 as e- acceptor

Diversity w/ Lactic Acid

• Diversity of products such as cheese or alcohol would not be posible

NAD, FAD, Heme in electron transport chain (ETC)

• Used to generate ATP
• Glucose and energy from Krebs cycle enter complex I to turn NADH to NAD and is facilitated by flavin to give an e- to Fe/S and to spit 4 H into the environment
• Complex II turns FAD into FADH to remove the 2 H from quinone so it can repeat its cycle
• ATP Synthase phosphorylates ADP into ATP through Oxidative Phosphorylation and make ATP

Oxygen environments

The rate of ATP depends on the respiration and nutrient availablity and if the bacteria can be sustained w/ the respiration type

• O2 has the terminal electron Acceptror and it has the fastest ATP and growthrate
• No O2 w/terminal for the fastest growth rate Without Oxygen, organic molecules in the acceptor, makes slower ATP and growth rate. This leads to decreased synthesis of the ATP and a reduced cell growth.

Specific Survival Characteristics

Survival and growth of bacteria and metabolic and physical characteristics all depend on bacterial characteristics.

• Different ATP synthesis relies on how much energy in a reaction (low to high) from the environment organic < inorganic < oxygen

Temperature Factors on Binary Division

When too cold, no growth, but instead a membraned gel Too hot- Protein and enzymes will denature until a membrane fails an ends with cell

Minimum, Optimal, & Maximum

• Minimum: The temperature below which there is no growth.
• Optimal: The temperature at which the growth rate is maximum.
• Maximum: The temperature above which there is no growth.

Decimal Reduction Time

• Decimal Reduction Time (D value): Time to reduce viable (living cells).

Oxygen & Bacteria Growth

• Obligate aerobe- must have oxygen to grow
• Microaerophile- only grow in low O2 conditions
• Facultative- aerobes/anaerobes prefer O2 to grow if available but can use other pathways if no O2 is available
• Obligate anaerobe- only grow without O2
• Aerotolerant anaerobe- grow with O2 but doesn’t use O2

Sodium

• Sodium contains gradient and nutrients with a lower amount of bacteria

Questions cover methods to measure the inhibition of bacterial DNA replication, identification of bacterial growth phases and impact of molecules on bacterial gene expression. Techniques for gene expression analysis, and bacterial isolation methods are discussed.