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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
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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
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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
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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)
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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
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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
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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.
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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
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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
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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
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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
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Define chemotaxis
Movement toward or away from chemicals (chemoaffectors)
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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)
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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)
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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
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Describe the proteins involved in chemotaxis
- CYTOPLASMIC PROTEINS
- CheR: methylation
- CheB: demethylation
- CheW – aids autophosphorylation of CheA
- CheA: sensor, histidine kinase
- CheY: response regulator
- CheZ: phosphatase, dephosphorylation
- MEMBRANE PROTEINS
- MCPs (methyl-accepting chemotaxis proteins):
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How do bacteria talk to eachother?
- Using signaling molecules (quorum sensing)
- Via cell-cell contact
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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
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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
- *ALLOWS INTERSPECIES COMMUNICATION
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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
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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)
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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 developmentCells commit suicide to become the stalk while others differentiate into myxospheres to be released/germinate to form a new colony when resources are sufficient
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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
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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
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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)
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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
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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
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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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