1. What are the domains and their characteristics?
    • Bacteria: Peptidoglycan in cell wall
    • 70S (30S/50S) ribosomes
    • Formyl Methionine initiator
    • No introns in RNA
    • no histones/nucleosomes
    • smaller RNA polymerase than Archaea/eukarya
    • not sensitive to diptheria toxin
    • Archaea: Pseudopeptidoglycan in cell wall
    • 70S (30S/50S) ribosomes - still not sensitive to antibiotics that affect prokaryotes
    • introns in RNA
    • histones/nucleosomes
    • sensitive to diptheria toxin
    • Eukarya: 80S (40S/60S) ribosomes
    • introns in RNA
    • histones/nucleosomes
    • sensitive to diptheria toxin
  2. Define the Archaea and the 3 groups contained within it
    • Methanogens: Obligate anaerobes
    • produce methane from CO2 or acetate
    • Inhabit groundwater, hot springs, swamps, sewage
    • Halophiles: Require high salt concentrations (3-5M)
    • Have chloride pumps to maintian osmotic stability
    • Have proton pumps to create proton potential for ATP synthesis
    • Found in salt lakes
    • Thermophiles: Growth between 55-100C
    • Most are anaerobic
    • Most are sulfur-dependent
    • Found in hot sulfur springs, hydrothermic vents
  3. List and describe the structures of a typical bacterial cell and their function (don't include cytosolic components)
    • Flagella: motility
    • Role in virulence of pathogens
    • Fimbriae: type of pili, look like tiny hairs
    • Aids in adherance to surfaces
    • Increase pathogenicity by allowing attachment to host cells
    • Sex pili: Required for conjugation and transfer of F plasmid
    • Found exclusively in gram negative
    • Glycocalyx: extracellular polysaccharide/polypeptide layer
    • Slime layers (biofilm) and capsules
    • Adherance
    • Protection from dessication (retains moisture)
    • Protection from phagocytosis
    • Carbohydrate reserve
    • Resistant to antibodies and drugs
    • Increase virulence
    • Cell wall: surrounds cell
    • Protects cell from osmotic lysis
    • Gives cell its morphology
    • Gram positive - thick peptidoglycan, thin periplasmic space, no outer membrane/LPS, purple in gram stain
    • Gram negative - thin peptidoglycan, large periplasmic space, outer membrane/LPS, pink in gram stain
    • Periplasm: cytochrome C location (ETC)
    • Breaks down nutrients into smaller molecules
    • Detoxifying agents
    • Transports of solutes through outer membrane (in/out)
    • Osmotic regulation
    • Cell membrane: Phospholipid bilayer + proteins
    • Permeability dependent on size/hydrophobicity/charge of molecule
    • ATP synthesis (location of ETC)
    • Solute transport
    • Protein secretion
    • Syntehsis of cell wall components
    • Signaling to other cells
    • Osmotic regulation (aquaporins)
  4. List and describe the structures of a typical bacterial cell and their function (cytosolic components)
    • Cytoplasm: Viscous
    • Contains proteins, salts, metabolites, DNA inclusions, etc
    • Cytosol is solube portion
    • Intracytoplasmic membranes: where specialized physiological activities occur (methane oxidation, nitrite oxidation, nitrogen fixation, photosynthesis)
    • Not found in all bacteria
    • Gas vesicles: fill with air to adjust buoyancy
    • Chlorosomes: contain light harvesting pigments
    • In photosynthetic bacteria
    • Magnetosomes: naviational device inside magnetotaxic bacteria
    • Granules: used for storage of starch, fat, sulfur, or phosphate
    • Nucleoid: area for DNAPlasmids: Can give new traits (antibiotic resistance)
    • Not present in all bacteria
    • Endospores: Dormant structures formed under adverse conditions
    • Highly resistant
    • Preservation mechanism
    • Cytoskeleton: important in maintaining cell shape
    • Fts Z - recruits other proteins to form septal ring during division (VERY IMPORTANT)
    • Similar to tubulin
    • Mre B - Helps determine shape of NON-SPHERICAL cells
    • Aids in cell polarity, chromosome segregation
    • Not found in spherical cells
    • Similar to actin
    • Crescentin: Responsible for viroid shape
    • Mutants are rod shaped
    • Similar to intermediate filaments
  5. Flagella Structure in depth
    • Huge protein complex, 4 parts
    • Filament: hollow
    • Made of flagellin
    • Extends to exterior of cell
    • Propels and pushes cell forward when it's rotated
    • Hook: connects filament to cell
    • Basal Body: embedded within the membrane
    • Anchors flagellum to cell wall
    • Consists of MS, C, P, and L rings (Gram positive do not have P or L rings) and a central rod
    • Motor: lies at base
    • Causes rotation
    • Consists of stator (for energy) and rotor (for rotation)
  6. Describe how bacteria reproduce
    • Binary fission (asexual process)
    • Generation time is the time required for a population to double
  7. List and describe how bacterial growth is measured (general)
    • Measurement of cell number (or cell mass) over time
    • Detected as colonies on solid media, or turbidity in liquid media
    • Direct measurement: counting individual cells
    • Indirect measurement: Analysis of specific cellular components (Protein, DNA, RNA, etc) or determination of cellular activities (O2 consumption, CO2 production, etc)
  8. Specific methods to measure growth
    • Turbidimetry: Indirect measurement
    • Measure of light absorbance by solution
    • Simplest and most rapid method
    • Proportionality between OD (Optical Density) and cell denisty only exists for OD < .4 [dense samples must be diluted]
    • Viable Cell Count: Direct measurement
    • counts only living cells
    • Each cell gives rise to a colony
    • Serial dilution created before spread plating
    • Underestimates number of live cells due to clumping, inefficient single-cell growth
    • Total cell count: Direct measurement
    • Petroff Huaser chamber
    • Count individual cells under microscope using special slides
    • Does not distinguish between live and dead cells
    • Cannot be used on low density cultures
    • Coulter Counter / Flow Cytometer
    • Bacteria pass through narrow pore producing pulse
    • #pulses = #cells
    • Can measure size of cells (proportional to pulse magnitude)
    • Dry weight: indirect measurement
    • Harvest, dry, and weigh cells
    • total weight / individual weight = # cells
    • Measure Cellular components over time: indirect measurement
    • Protein, DNA, RNA measured against a standard curve
  9. What are the four phases of bacterial growth?
    • Lag phase: Occurs when cells in stationary phase are transferred to fresh media
    • Period of adjustment and preparation for grwoth
    • No increase in number, but may increase in size
    • Log phase: Exponential growth occurs
    • Balanced growth (all components grow at same rate)
    • Cells VERY VULNERABLE to adverse conditions
    • Stationary phase: No net growth
    • Overall population stabilizes
    • More resistant to adverse conditions (all E put toward survival, not growth)
    • Death phase: Cells die exponentially
    • Total # of viable cells decreases
    • Typically a few cells will survive or form endospores/cysts
  10. Adaptive responses of bacteria to nutrient limitation (starvation)
  11. Macromolecular composition changes as a function of growth rate
    • Faster growing cells have...
    • a higher ration of RNA to protein ratio 
    • Because polymerization of AA is constant (more ribosomes needed [rRNA] and mRNA)
    • a larger mass
    • initiation of DNA replication requires a minimum cell mass per replication origin
    • Faster growing cells must have multiple replication origins
    • more DNA
    • DNA replication takes 60 minutes
    • cells with generation time <60m are born with partially replicated DNA
    • Multiple replication origins
  12. Environmental requirements for growth
    • Temperature
    • Aeration
    • pH
    • Minimal media (M-9): slower growth, smaller cells
    • Rich (complex) media: faster growth, larger in mass
    • Less energy is diverted to making 'building blocks'
  13. What is diauxic growth?
    • Preferential growth on one carbon source BEFORE growing on a second
    • ex- E. coli consuming all glucose before using lactose
  14. What is catabolite repression by glucose?
    • Glucose inhibits synthesis of enzymes used in catabolism of non-primary carbon sources
    • Very common in bacteria
    • Exceptions in Rhizobium and P. aeruginosa which prefer organic acids over glucose
  15. Adaptive responses of bacteria to nutrient limitation
    • Changes in cell size: become smaller
    • reductive division (division with no growth)
    • Dwarfing - continuous cell size reduction after reductive division
    • Self-digesting process
    • Morphological changes: rod-shape to coccoid
    • Changes in surface properties: surface becomes hydrophobic and adhesive
    • Allows cells to aggregate and adhere to adsorbed nutrients on surfaces
    • Changes in metabolic activities: rate slows
    • Turnover of proteins and RNA to serve as energy sources
    • Changes in protein composition: specific genes turned on (survival proteins)
    • Changes in resistance to environmental stress: more resistant to high temp, osmotic stress, harsh chemicals
    • Control of rRNA synthesis: starvation slows rRNA synthesis (lack of AA)
  16. What is growth yield?
    • Efficiency of an organism in converting nutrients to cell mass
    • Weight biomass produced per weight nutrient consumed
    • Measured by comparing to a growth yield constant
    • Can also be expressed in terms of energy yield
  17. Growth kinetics
    • Equation for exponential growth: logN = k(t-t0)/2.303 + log N0
    • N = # cells/unit volume, t = time at end (hr), K = specific growth rate constant/hr
    • Relationship between generation time and growth rate: .693 = kg
    • g = generation time, k = growth rate constant
    • If a nutrient is limited it will limit growth rate
    • Yield is proportional to [limiting nutrient]
    • Yield is not affected by temp, just rate
    • Growth rate is a function of substrate concentration only at very low [S]
    • Dependent on temperature, pH

    Determining # of cells after a given time. N = N0 x 2n (n = # generations)
  18. What is synchronous growth? Steady state growth?
    • Synchronous: all cells at the same stage of the cell cycle
    • Steady state: balanced growth, all components maintained at a constant ratio (log phase)
  19. Info about chemostat
    • Chemostat: apparatus for continuous exponential growth of bacteria
    • Fresh medium continuously supplied to growth chamber at a certain growth rate (F)
    • Limiting nutrient purposely kept at a controlled level to allow for manipulation of growth rate
    • Continuous removal of cells and nutrients to maintain a constant volume
  20. Relationship between dilution rate and growth rate constant
    • Dilution rate: D = F/V
    • F = flow rate, v = volume of media in growth chamber
    • At steady state, K = D
    • k = growth rate constant, D = dilution rate
  21. Relationship between cell mass and [substrate]
    • Y = growth yield constant = mass of cells per amt nutrient used up
    • Y=x/(Sr-S)
    • Nutrients are substrate
  22. What are the two "periods" in the bacterial cell cycle?
    • C period: DNA replication occurs
    • D period: Cell division
    • Chromosome separation
    • Mother cell splits to form two daughter cells
  23. What are the steps in bacterial cell division?
    • Cell increases in mass
    • DNA replication occurs when critical mass is reached: Initiation -> replication -> chromosome partitioning -> chromosome separation
    • *multiple rounds of DNA replication occur simultaneously
    • Cytokinesis: Septum formation
    • g neg - septation and cell separation occur simultaneously
    • g pos - septation occurs before separation (chains of cells as result)
  24. What proteins are associated with septal ring formation and cell division?
    • fts genes: encode the proteins that form the contractile ring (Z ring)
    • FtsZ: assembles all the fts proteins to the z ring
    • FtsA: hydrolizes ATP and directly interactions with FtsZ
    • ZipA: anchors the FtsZ ring to the membrane (no ZipA, no cleavage)
    • EnvC: outer membrance constriction/separation of cells
    • EnvA: necessary for cell separation (lipid A synthesis)
    • Constriction begins at septal site after all proteins are in place
  25. How is the site of septum formation determined?
    • Nucleoid occlusion system: nucleoid mass prevents formation of septal ring in the regeion it occupies
    • Septal ring can only form at polar ends or between the two nucleoids
    • Min system: Prevents formation of FtsZ at cell polls (forces it to center)
    • Consists of MinC, MinD, and MinE
    • MinC - interferes with polymerization of FtsZ
    • MinD - ATPase for MinC
    • MinE - determines location for MinCD, causes them to oscilate between the two halves of the cell
  26. Describe the Meselson-Stahl experiment
    • Showed DNA replication is semiconservative
    • Cells were grown in 15N
    • DNA was isolated and centrifuged, showed one band at 15N
    • Cells were transferred to 14N media
    • After one generation of growth there was a small band at 14N and a thick band at 15N
    • After many generations of growth almost all the band was at 14N
  27. Describe the nature of the DNA molecule
    • Double strand structure
    • Strands are complementary and antiparallel
    • A=T C=G
    • Deoxyribose bonded by a phosphodiester bond
    • Semiconservative replication
    • Right handed helix (due to hydrophobic interaction of bases)
    • Nucleoid is highly condensed
    • + supercoiling: overwound, twisted in same direction as helix
    • - supercoiling: relaxed, twided in opposite direction as helix
    • Supercoiling becomes a problem as replication fork proceed (+ supercoiling occurs in DNA ahead of replication fork)
    • DNA gyrase must relieve this strain
  28. Enzymes involved in DNA replication
    • DNA gyrase: removes positive supercoiling in unreplicated DNA
    • Binds to both strands, makes a double strand bread, pulls one strand through, reseals break
    • DnaB: helicase, unwinds DNA
    • DnaA and C: involved in initiation complex
    • SSB protein: bind to/keep DNA single stranded
    • Primase: makes RNA primers
    • DNA pol I: primer removal, filling in of gaps
    • DNA pol III: DNA synthesis (needs 3' OH end to add nucleotides)
    • Ligase: forms phosphodiester bond between 5'-P and 3'-OH
  29. What is a replisome?
    • "Replication factory"
    • Unreplicated DNA threads through stationary replication forks (bidirectioinal) 
  30. DNA replication process
    • Initiation: 30 proteins initiate DNA replication
    • DnaA - binds to Origin Replication
    • DnaB (helicase) - unwinds strands at OriC
    • SSB - keep DNA single stranded
    • Extension: Synthesis is 5'-3' ONLY (requires a 3'-OH end)
    • RNA primer needed to intiate replication
    • DNA Pol I and DNA Ligase attach okazaki fragments together
    • Termination: occurs at Ter sites
    • Multiple ter sites (think traffic lights)
    • Force replication to a single direction
    • Separation: detachment of DNA molecules from eachother
    • segregation not well understood
    • theories - replisome? protein? protein tether to membranes?
  31. (transcription) What are RNA polymerase, polycistronic mRNA, sigma factors?
    • Polycistronic mRNA: encode for many proteins
    • RNA polymerase: carries out transcription
    • Occurs 5' -> 3' producing a ssRNA molecule
    • Does not require a primer
    • Can unwind DNA helix (doesn't need helicase)
    • Binds to sigma factor to be active
    • Sigma factors: recognize promotor sequences
    • Diff sigma factors recognize diff sequences
    • sigma70 - main sigma factor in E. coli
    • sigma54 - transcription of genes activated by stress condition, growth transition, and morphological changes
  32. Stages of RNA synthesis
    • Initiation: begins at promotor region
    • RNAP binds to promotor (guided by sigma factor)
    • Recognition of a specific sequences (~10 upstream and ~35 upstream)
    • Sigma factor is released once RNAP passes the promotor region
    • Chain elongation: new ribonucleotides added to growing chain
    • Must add to 3' OH end
    • DNA gyrase uncoiles DNA downstream
    • Termination (Factor-independent): does not require a protein factor
    • End of RNA contains complementary sequence of bases that hybridizes to form termination hairpin loop
    • Termination (Factor-dependent): requires protein factors to dissociate RNA-DNA hybrid (Rho, Tau, or NusA)
    • Rho - ATP dependent RNA/DNA helicase
    • Disrupts the duplex at the end of transcription, removes RNAP from DNA, and reforms DNA-DNA duplex
  33. Define promoter strength and discuss how it is related to gene expression
    • Promoter strength: influences frequency of transcription
    • Srong have initiation every few seconds
    • Weak have initiation every few minutes
    • Transcription factors: bind near promotor and affect the trascription frequency
    • Can be + or -
  34. What are the products of transcription?
    • mRNA: translated into polypeptides
    • tRNA: smallest RNA, carries AA to ribosome
    • rRNA: structural RNA that makes up ribosome
    • tRNA and rRNA require additiional processing
  35. Mechanisms of regulation of transcription
    • Control the levels of gene expression
    • Transcription factors: bind to sequences of DNA to affect binding of RNAP to promotor
    • Can regulate several operons (regulon)
    • Activators: positive regulators
    • Bind upstream of promotor at enhancer sites
    • Promote RNAP binding
    • Repressors: negative regulators
    • Bind downstream of promoter, blocking RNAP
  36. Trp operon regulation
    • Repressor with corepressor (trp) binds to operator (activated by trp)
    • Attenuation: regulates transcription after it has started but before first gene is transcribed
  37. Stages of protein synthesis
    • Initiation: formation of initiation complex (30S, mRNA, fMet-tRNA, and initiation factors)
    • Initiator tRNA carrying formylmethionine binds to start site of mRNA at P site on ribosome (at Shine-Dalgamo sequence)
    • 50S portion binds (formation of 70S complex)
    • Elongation: begins as an aminoacylated tRNA binds to A site
    • Peptide bond forms between first/second AA (peptidyltransferase)
    • first tRNA is released
    • Second tRNA moves to P site
    • A site is now available for next charged tRNA
    • Termination:occurs at stop codon (UAA, UGA, UGG)
    • Requires specific proteins (release factors)
    • Attacks H2O on link between tRNA and peptide
    • 70S ribosome is dissassembled
  38. What is tRNA charging?
    • aminoacy-tRNA synthetase attaches amino acid to tRNA
    • The tRNA is now "charged"
  39. Explain the role of chaperones in protein folding, the types of chaperons
    • Information for folding is contained within the AA sequence, but chaperones assist
    • Prevent misfolding and aggregation
    • Ensure rapid folding
    • DnaK-DnaJ-GrpE: bind to polypeptides still on the ribosome
    • Prevent premature folding / aggregation of protein during synthesis
    • GroEL / GroES, the GroE system: Bind to polypeptides after release from ribosome and assist with proper folding
  40. Inhibitors of protein synthesis (in bacteria)
    • Erythromycin: inhibits translocation
    • Streptomycin: inhibitis initiation and causes mistkaes in reading mRNA at low concentrations
    • Tetracycline: inhibits bind of aminoacyl-tRNA
    • Chloramphenicol: Inhibits polytransferase
    • Puromycin: resembled charged tRNA and causes premature chain termination
Card Set