BactPhysFinal

Involves a histidine kinase that (1) autophosphorylates in response to a signal, then (2) transfers phosphoryl group to a response regulator which regulates transcription

• 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

• 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

• 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)

• 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

• 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

• 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.

• 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

• 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

• Draw linear line through data
• Generation time: time it takes OD to double
• Growth rate: .693/Generation time
• *convert G.rate to hours

• 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

Movement toward or away from chemicals (chemoaffectors)

• 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)

• 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)

• 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

• 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):

• Using signaling molecules (quorum sensing)
• Via cell-cell contact

• 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

• 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

• Cell divides to produce 2 daughter cells that acquire phenotypic properties that DIFFERENTIATE them from a precursor cell 
• Can be morphological or physiological

• 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)

• 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

• 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

• 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

• 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)

• 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

• 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

• 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