1. What is a two-component system and how does it work (generally)?
    Involves a histidine kinase that (1) autophosphorylates in response to a signal, then (2) transfers phosphoryl group to a response regulator which regulates transcription
  2. List and define the 3 types of proteins of the two-component signaling systems
    • Histidine kinase: A sensor component receives/transmits signal to RR protein
    • Response regulator: regulatory protein that receives signal from HK and transmits to target
    • Phosphatase: inactivates RR-P by dephosphorylation
  3. Describe anaerobiosis (general)
    • Anaerobiosis: shift from aerobic to anaerobic environment 
    • 1. Metabolic changes
    • Citric acid cycle is replaced by reductive TCA (noncyclic)
    • NADH no longer produced (no need to regen NAD+)
    • 2. Induction of anaerobic genes
    • Electron acceptor synthesis regulated based on availability
    • +O2 represses synthesis of anaerobic reductase
    • +NO3- represses synthesis of other reductases
  4. Describe the ARC system in detail
    • anaerobic respiratory control OR anoxic redox control system
    • Arc B: transmembrane sensor kinase
    • Activated by anoxia (increase in reduced electron carriers in cell [NADH], anaerobic metabolite buildup [pyruvate, lactate]
    • Arc A: response regulator
    • Represses genes for aerobic growth
    • Induction of genes for cytochrome d oxidase (high O2 affinity at low O2 levels)
  5. Describe the FNR system in detail
    • Fumerate Nitrate Reductase system
    • Global regulator protein activated during anaerobic growth
    • + transcription of anaerobic growth genes
    • - transcription of aerobic growth genes
    • may regulate the same genes as ARC system
    • Has DNA binding ability
    • Iron-Sulfur center (reduced/oxidized based on O2 levels)
    • Oxidized: inactivated
    • Reduced: activated
    • binds to DNA to inhibit aerobic genes
    • binds to DNA to induce anaerobic genes
  6. Describe the PHO regulon in detail
    • Regulon: set of nontcontiguous operons controlled by a common regulator
    • PHO regulon
    • contains genes for regulation of phosphate assimilation when phosphate supply is limited
    • Under low phosphate conditions stimulates at least 38 genes for phosphate uptake
    • PtsS: periplasmic binding protein
    • Forms repressor complex if P is bound
    • PhoR: histidine kinase/phosphotase
    • Detects P, can activate or deactivate PhoB
    • PhoB: response regulator
    • positively regulates PHO regulon when active
  7. How do bacterial cells protect themselves from osmotic stress?
    • Bacteria need to adjust the amount of solutes within the cell to match that outside the cell
    • Regulate outer membrane proteins (porins)
    • OmpC: small pore
    • OmpF: large pore
    • Hypteronic/Hot: increased OmpC (retards inflow of solutes)
    • Hypotonic/Cold: increased OmpF (increase inflow of dilute nutrients)
    • Lakes/streams
    • Enz/OmpR two component system is regulator.
  8. Describe EnvZ/OmpR system in detail
    • EnvZ: inner membrane histidine kinase
    • osmotic sensor
    • transmembrane; periplasm and cytoplasm
    • OmpR: Response Regulator
    • Cytoplasmic protein
    • Hypertonic: OmpR phosphorylated
    • stimulates transcription of OmpC (stimulates kinase activity of EnvZ)
    • represses ompF byinducing micF (antisense RNA that prevents OmpF mRNA expression)
    • Hypotonic: OmpR unphosphorylated
    • stimulates transcription of OmpF
  9. Explain the relationship between absorbance and cell number
    • Simplest and most rapid method for cell counts
    • Light is passed through solution, amount of light scattered is proportional to number of cells (< .4 ONLY)
    • Dense samples must be diluted.
    • Counts both living AND dead cells
  10. How to determine growth rate and generation time based on bacterial growth curves?
    • Draw linear line through data
    • Generation time: time it takes OD to double
    • Growth rate: .693/Generation time
    • *convert G.rate to hours
  11. Compare M9 and NB for lab 3
    • NB: grows faster, larger cell, more protein per cell, more RNA per cell
    • M9: slower growing, smaller cell, less protein per cell, less RNA per cell
  12. Define chemotaxis
    Movement toward or away from chemicals (chemoaffectors)
  13. How do bacteria respond to attractants and repellents via the two-component system?
    • CheA: cytoplasmic histidine kinase
    • CheY: response regulator
    • Signal sensed by MCP at surface of cell, transduced through cytoplasmic signaling pathway, leads to autophosphorylation of CheA
    • Attractants decrease rate of autophosphorylation
    • Repellents increase rate of autophosphorylation
    • CheY-P increases tumbling frequency (CW)
  14. Explain how flagellar movement responds to chemical gradients
    • High attractants increase running (CCW)
    • Attractants increase methylation
    • reduce level of autophosphorylation
    • reduce level of CheY-P
    • Reduced tumbling (increased swimming)
    • High repellent increase tumbling (CW)
    • Repellents decrease methylation
    • Increase level of autophosphorylation
    • Increase level of CheY-P
    • Increased tumbling (reduced swimming)
  15. Give examples of attractants and repellents
    • Attractants
    • 1. Amino acids -  serine, alanine, glycine, cysteine, threonine
    • 2. Sugars - glucose, maltose, galactose, ribose
    • Repellents
    • 1. Valine – leads to starvation of isoleucine (same pathway for making valine) -> stringent response is elicited
    • 2. H2S, indole, acetate – metabolites that indicate crowding
    • 3. Certain fatty acids – indicates low pH
    • 4. Certain alcohols – dries out membrane
  16. Describe the proteins involved in chemotaxis
    • CheR: methylation
    • CheB: demethylation
    • CheW – aids autophosphorylation of CheA
    • CheA: sensor, histidine kinase
    • CheY: response regulator
    • CheZ: phosphatase, dephosphorylation
    • MCPs (methyl-accepting chemotaxis proteins):
  17. How do bacteria talk to eachother?
    • Using signaling molecules (quorum sensing)
    • Via cell-cell contact
  18. Why do bacteria talk to eachother? What processes can it affect?
    • Allows coordination of behavior to respond quickly to survive environmental changes
    • Adaptation to availability of nutrients
    • Defense against other microbes (biofilm)
    • avoidance of toxic compounds (biofilm)
    • Initiate development
    • expression of certain genes
    • Processes related...
    • Bioluminescence
    • Myxobacteria fruiting body formation
    • Sporulation in B. subtilis
    • Biofilm formation
    • Conjugal plasmid transfer
    • Activation of virulence genes
  19. Explain quorum sensing.  Types of molecules?
    • Each species synthesizes a specific signal molecule that can freely diffuse across the membrane
    • These behave like effector molecules and will bind to regulators changing gene expression OR cell behavior
    • Types of molecules include
    • Oligopeptide pheromones: small peptides typically found in gram positive bacteria
    • Bind to surface and cause cascade to a cytoplasmic receptor (enzyme or transcription factor)
    • AHLs (acelated homoserine lactones)
    • AHL1: found in gram negative bacteria
    • first identified in bioluminescent Vibrio
    • AHL2: found in gram neg/pos
  20. Define bacterial development
    • Cell divides to produce 2 daughter cells that acquire phenotypic properties that DIFFERENTIATE them from a precursor cell 
    • Can be morphological or physiological
  21. Why do bacteria undergo development/differentiation?
    • Generation of "resting" cell forms more resistant to environmental stresses (endospores, myxospores)
    • Generation of cell forms specifically for dispersal of bacteria (swarmer cells)
    • Generation of cell forms performing specific functions (heterocysts)
    • Generation of cell forms to establish symbiotic relationship (nitrogen-fixing root nodules)
  22. What induces development in myxobacteria?
    • Two life stages
    • vegetative: abundance of nutrients
    • Cooperative feeding
    • Swarms move and feed together on other microbes
    • developmental: food supply is depleted
    • myxobacteria aggregate into "fruiting bodies"
    • Starvation is the signal for development
    • Cells commit suicide to become the stalk while others differentiate into myxospheres to be released/germinate to form a new colony when resources are sufficient
  23. Explain the intercellular signaling mechanism involved in multicellular development
    • Development involves two signal molecules
    • A-signal: detects starvation and cell density in population
    • Ensures fruiting body formation is initiated on when a suffiently high # of cells are starving and cell density is sufficient for formation (millions of cells)
    • Induces cells to come together
    • C-signal: contact-mediated signal
    • Guides formation of fruiting body (avoids traffic jams by forcing cells to move end to end)
    • Induces sporulation
    • Directions efficient feeding during predation
  24. Describe the process of sporulation (general)
    *might not need to know
    • Forms spores in response to nutrient deprivation/harsh conditions
    • Stage 1: axial filament formation (unequal, 1/4 cell size)
    • Stage 2: septum and prespore formation (unequal, 1/4 cell size)
    • Stage 3: forespore development (tiny spore WITHIN cell
    • Stage 4: cortex formation (tiny spore within cell becomes more developed)
    • Stage 5: coat formation (tiny spore within cell gets coat)
    • Stage 6: maturation
    • Stage 7: release of mature spore
  25. What drives vegetative cells to undergo sporulation?
    • Favorable conditions: endospore-forming bacteria undergo vegetative growth
    • Unfavorable conditions: 
    • Chemotaxis - increase chances of finding nutrients
    • Antibiotic synthesis - inhibit competing microbes
    • Secretion of degredative enzymes - breakdown potential food
    • Development of competence - allow uptake of exogenous DNA
    • *SPORULATION IS A LAST RESORT TO SURVIVE (vegetative cell dies)
  26. Describe sporulation specific to B. subtilis
    • spo genes named after the stage they block and a letter to indicate variant (spo0A do not proceed to stage 1)
    • Sporulation genes are differently expressed in mother cell and endospore, resulting in 2 types of cells.
    • SpoA: master regulator of sporulation genes, activated by nutrient limitation
    • 10 different sigma factors in B. subtilis, each directs RNAP to a different set of promoters
    • Sporulation regulated by a phosphorelay signal transduction system in response to environmental, metabolic, and cell cycles
  27. Describe the phosphorelay involved in sporulation
    • The two most important kinases (A and B) for sporulation: sensor kinases that phosphorylate Spo0F (response regulator)
    • Spo0F: (phosphorelay protein) accepts phosphate from the activating kinases, serves as a substrate for the Spo0B protein
    • Spo0B: (phosphorelay protein) transfers phosphate from Spo0F to Spo0A producing Spo0A-P
    • Spo0A-P: regulates expression of genes in Spo0A regulon (phosphorylation activates transcription functions)
    • Increased levels of Spo0A-P stimulate axial filament formation, polar septation, and genes for the forespore/mother cell exclusively
  28. What is fratricide?  Why would B. subtilis want to do this?
    • Fratricide: killing of siblings
    • Occurs before sporulation
    • cells are killed and used as nutrients in an attempt to avoid sporulation
    • Nutrient starvation induces Spo0A in 1/2 the cells which causes toxin secretion and death to the Spo0A OFF cells.  When all Spo0A OFF cells are digested the remaining population will form spores
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