MICR 221 - Midterm 2 Exam With
Complete Solutions
Bacterial motility and taxis - ANSWER - Many bacteria are motile (move
independantly by using flagella or pilli)
- Taxi: directed movement in response to a stimuli
- Positive taxis: movement towards a stimulus (nutrients)
- Negative taxis: movement away from stimulus (toxic substances)
- Chemotaxis: move towards or away from attractants/repellents
Flagella - ANSWER - Long helical structures (20nm diameter, up to 20 um long)
- Mainly used for motility in liquid environments
- Flagellar swimming results from rapid rotation of flagella (flagella rotate 100-
1000 times per second, can swim up to 100 um/s)
- Number and distribution vary
- Monotrichous: flagellum on one pole
- Atrichous: no flagella
- Peritrichous: flagella distributed over surface
Flagellum structure - ANSWER - Basal body
- Filament
- Hook
Basal body of flagellum - ANSWER - Attaches flagellum to cell envelope
- Contains motor (responsible for rotation)
- Protein structure composed of central rod, several rings
- Most gram negatives have: L, P and Ms rings embedded in the cell envelope
and C ring in cytoplasm.
- Export functions: basal body transports subunits during flagellum assembly
filament of a flagellum - ANSWER - Long helical structure that extends from
surface
- Rotation moves the cell
Hook of flagellum - ANSWER - Flexible, bent structure
- Transmits rotation from basal body to filament
Flagellar motors - ANSWER - Basal body is a motor:
- Rotor (rod, MS ring)
- Bushings/bearings (p,L rings)
- Stator (MotA, MotB)
,- Switch (C ring) determines the direction of rotation
- Rotation usually powered by proton motor force (proton channel: MotA, MotB)
Flagellum assembly - ANSWER - Basal body assembles first, then hook, then
filament
- Subunits exported up hollow core, added to the distal end
- Filament composed of thousands of flagellin proteins (assembled in repeating
helical pattern, incorporated under cap protein)
Flagella and the immune system - ANSWER - Flagella are major immune targets
(because they are exposed on the surface)
- Innate immune system can recognize flagellin (toll-like receptor 5 (TLR5))
- Binding of flagellin to TLRS activates NF-kB (transcription factor)
- Leads to production of pro-inflammatory cytokines
- Flagella are also targeted by the adaptive immune system:
- Major antigenic structure
- Each flagella is composed of 1000s of copies of protein subunits
- Antibody binding leads to phagocytosis
Immune Evasion and Phase variation - ANSWER - Once in host, some bacteria
stop producing flagella
- Bacteria don't want to be detected by immune system, once they enter the host
they may remodel or remove flagella to not be recognized
- Some bacteria alternate between different flagellins (phase variation -
reversible change to phenotype)
Chemotaxis - Random walk - ANSWER - If no chemical gradient, no benefit to
moving in a particular direction
- Bacteria will move in a random walk (swim forward (run), occasionally reorient
(tumble) and swim forward in new random direction (run))
Flagellum rotation: - ANSWER - Direction of flagellum rotation determines if cell
is running or tumbling
- Monotrichous bacteria (one flagellum): run (counterclockwise rotation), tumble
(clockwise rotation)
- Peritrichous bacteria (multiple flagellum): run (counterclockwise rotation),
tumble (clockwise rotation)
Chemotaxis: A biased random walk - ANSWER - In a gradient, some directions
are advantageous
- Move in biased random walk
- If direction is favourable: longer runs and fewer tumbles
- Unfavourable direction: tumbles more
- Favourable or unfavourable is determined based on concentration of attractant
or repellent
, Methyl-accepting chemotaxis proteins (MCPs) - ANSWER - Chemoreceptors in
cytoplasmic membrane
- bind attractant or repellent and initiate flagellar rotation
- Can sense different repellents or attractants
- By default flagella rotate counterclockwise (to favour runs)
- MCPs can signal to flagella to change directions (initiate a tumble)
- MCP ligand binding domain forms a complex with a ligand
- This ligand then impacts the comformation of MCP signalling domain
- CheW is bound to MCP signalling domain (presence of ligand impacts CheW
activity)
If no attractant ligand is bound to MCP... - ANSWER If direction is "bad", cell
tumbles to reorient:
1. CheW detects no attractant bound to MCP
2. CheW causes kinase CheA to be auto-phosphorylated
3. CheA-P phosphorylates CheY
4. CheY-P binds to flagellum switch, causing CW rotation
5. CheY-P dephosphorylates over time by CheZ
If attractant is bound to MCP... - ANSWER If direction is "good", cell favours run
(cell wants to keep moving forward)
- CheW detects attractant bound to MCP
- CheW does not cause CheA to be autophosphorylated
- CheY not phosphorylated, doesn't bind to flagellum switch (counterclockwise
rotation favoured)
excitation of flagellum - ANSWER - Ligand binding results in counterclockwise
bias (longer runs, fewer tumbles)
- Direction of rotation based on input from many MCPs (more MCPs with bound
ligand, less CheyY-P formed, greater counterclockwise bias)
adaptation of flagellum - ANSWER - Remember: ligand binding to MCP:
decreases CheA autophosphorylation, decreasing amount of CheY-P
- A few seconds after ligand binds, MCP is methylated by CheR
- Methylation of MCP: increases CheA autophosphorylation (more proteins to
interact with the switch) and increases amount of CheY-P
- Methylation offsets impact of ligand binding on flagellar rotation, removes
counterclockwise bias.
- If ligand leaves, methylated MCP still enhances CheA activity, which causes a
clockwise bias
Temporal gradients: - ANSWER - Bacteria usually measure concentration
gradients temporally not spatially (used to determine if they will swim up or
down a gradient)
- Current concentration - number of MCPs with ligand
- Past concentration - number of methylated MCPs
Complete Solutions
Bacterial motility and taxis - ANSWER - Many bacteria are motile (move
independantly by using flagella or pilli)
- Taxi: directed movement in response to a stimuli
- Positive taxis: movement towards a stimulus (nutrients)
- Negative taxis: movement away from stimulus (toxic substances)
- Chemotaxis: move towards or away from attractants/repellents
Flagella - ANSWER - Long helical structures (20nm diameter, up to 20 um long)
- Mainly used for motility in liquid environments
- Flagellar swimming results from rapid rotation of flagella (flagella rotate 100-
1000 times per second, can swim up to 100 um/s)
- Number and distribution vary
- Monotrichous: flagellum on one pole
- Atrichous: no flagella
- Peritrichous: flagella distributed over surface
Flagellum structure - ANSWER - Basal body
- Filament
- Hook
Basal body of flagellum - ANSWER - Attaches flagellum to cell envelope
- Contains motor (responsible for rotation)
- Protein structure composed of central rod, several rings
- Most gram negatives have: L, P and Ms rings embedded in the cell envelope
and C ring in cytoplasm.
- Export functions: basal body transports subunits during flagellum assembly
filament of a flagellum - ANSWER - Long helical structure that extends from
surface
- Rotation moves the cell
Hook of flagellum - ANSWER - Flexible, bent structure
- Transmits rotation from basal body to filament
Flagellar motors - ANSWER - Basal body is a motor:
- Rotor (rod, MS ring)
- Bushings/bearings (p,L rings)
- Stator (MotA, MotB)
,- Switch (C ring) determines the direction of rotation
- Rotation usually powered by proton motor force (proton channel: MotA, MotB)
Flagellum assembly - ANSWER - Basal body assembles first, then hook, then
filament
- Subunits exported up hollow core, added to the distal end
- Filament composed of thousands of flagellin proteins (assembled in repeating
helical pattern, incorporated under cap protein)
Flagella and the immune system - ANSWER - Flagella are major immune targets
(because they are exposed on the surface)
- Innate immune system can recognize flagellin (toll-like receptor 5 (TLR5))
- Binding of flagellin to TLRS activates NF-kB (transcription factor)
- Leads to production of pro-inflammatory cytokines
- Flagella are also targeted by the adaptive immune system:
- Major antigenic structure
- Each flagella is composed of 1000s of copies of protein subunits
- Antibody binding leads to phagocytosis
Immune Evasion and Phase variation - ANSWER - Once in host, some bacteria
stop producing flagella
- Bacteria don't want to be detected by immune system, once they enter the host
they may remodel or remove flagella to not be recognized
- Some bacteria alternate between different flagellins (phase variation -
reversible change to phenotype)
Chemotaxis - Random walk - ANSWER - If no chemical gradient, no benefit to
moving in a particular direction
- Bacteria will move in a random walk (swim forward (run), occasionally reorient
(tumble) and swim forward in new random direction (run))
Flagellum rotation: - ANSWER - Direction of flagellum rotation determines if cell
is running or tumbling
- Monotrichous bacteria (one flagellum): run (counterclockwise rotation), tumble
(clockwise rotation)
- Peritrichous bacteria (multiple flagellum): run (counterclockwise rotation),
tumble (clockwise rotation)
Chemotaxis: A biased random walk - ANSWER - In a gradient, some directions
are advantageous
- Move in biased random walk
- If direction is favourable: longer runs and fewer tumbles
- Unfavourable direction: tumbles more
- Favourable or unfavourable is determined based on concentration of attractant
or repellent
, Methyl-accepting chemotaxis proteins (MCPs) - ANSWER - Chemoreceptors in
cytoplasmic membrane
- bind attractant or repellent and initiate flagellar rotation
- Can sense different repellents or attractants
- By default flagella rotate counterclockwise (to favour runs)
- MCPs can signal to flagella to change directions (initiate a tumble)
- MCP ligand binding domain forms a complex with a ligand
- This ligand then impacts the comformation of MCP signalling domain
- CheW is bound to MCP signalling domain (presence of ligand impacts CheW
activity)
If no attractant ligand is bound to MCP... - ANSWER If direction is "bad", cell
tumbles to reorient:
1. CheW detects no attractant bound to MCP
2. CheW causes kinase CheA to be auto-phosphorylated
3. CheA-P phosphorylates CheY
4. CheY-P binds to flagellum switch, causing CW rotation
5. CheY-P dephosphorylates over time by CheZ
If attractant is bound to MCP... - ANSWER If direction is "good", cell favours run
(cell wants to keep moving forward)
- CheW detects attractant bound to MCP
- CheW does not cause CheA to be autophosphorylated
- CheY not phosphorylated, doesn't bind to flagellum switch (counterclockwise
rotation favoured)
excitation of flagellum - ANSWER - Ligand binding results in counterclockwise
bias (longer runs, fewer tumbles)
- Direction of rotation based on input from many MCPs (more MCPs with bound
ligand, less CheyY-P formed, greater counterclockwise bias)
adaptation of flagellum - ANSWER - Remember: ligand binding to MCP:
decreases CheA autophosphorylation, decreasing amount of CheY-P
- A few seconds after ligand binds, MCP is methylated by CheR
- Methylation of MCP: increases CheA autophosphorylation (more proteins to
interact with the switch) and increases amount of CheY-P
- Methylation offsets impact of ligand binding on flagellar rotation, removes
counterclockwise bias.
- If ligand leaves, methylated MCP still enhances CheA activity, which causes a
clockwise bias
Temporal gradients: - ANSWER - Bacteria usually measure concentration
gradients temporally not spatially (used to determine if they will swim up or
down a gradient)
- Current concentration - number of MCPs with ligand
- Past concentration - number of methylated MCPs
