-
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
-
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
-
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)
-
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
-
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)
-
Describe how bacteria reproduce
- Binary fission (asexual process)
- Generation time is the time required for a population to double
-
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)
-
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
-
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
-
Adaptive responses of bacteria to nutrient limitation (starvation)
-
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
-
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'
-
What is diauxic growth?
- Preferential growth on one carbon source BEFORE growing on a second
- ex- E. coli consuming all glucose before using lactose
-
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
-
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)
-
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
-
Growth kinetics
- Equation for exponential growth: logN = k(t-t0)/2.303 + log N0N = # 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 = N 0 x 2n (n = # generations)
-
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)
-
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
-
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
-
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
-
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
-
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)
-
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
-
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
-
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
-
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
-
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
-
What is a replisome?
- "Replication factory"
- Unreplicated DNA threads through stationary replication forks (bidirectioinal)
-
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?
-
(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
-
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
-
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 -
-
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
-
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
-
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
-
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
-
What is tRNA charging?
- aminoacy-tRNA synthetase attaches amino acid to tRNA
- The tRNA is now "charged"
-
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
-
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
|
|
