326R
Chemotaxis
Flagella
Bio
Monotrichous (one flagella) or peritrichous, atrichous, etc Can rotate clockwise or counterclockwise E. coli is multitrichous, but the flagella are all located at one end of the bacterium. The direction of the spin of the flagella controls movement
CCW: forward
CW: tumble
Chemotaxis
How does E. coli know where to swim?
Chemotaxis
In a liquid medium, E. coli shows long straight runs followed by short tumbles, followed by another run in a different, random direction.
This is called a random walk. E. coli runs about 95% of the time, tumbles 5% This applies to any media whether high or low in attractants or repellants, the bacterium will maintain the 95:5 ratio of running to tumbling If the concentration of an attractant is increased, the tumbling frequency is decreased (or the runs are longer). Decreasing concentration of attractants causes tumbling frequency to increase (i.e. the runs are shorter) Repellants work the same way but in reverse After a short time at the new concentration of attractant, the tumbling frequency re-sets to the baseline level (95:5) This is called a biased random walk. In liquid media there is no consistent gradient. In solid media, there is no motility.
How to get from A to B with only random changes in direction?
Experimentally, this kind of behavior can be seen in semi-solid media.
Chemotaxis
Methyl Accepting Chemotaxis Proteins (MCPs)
Binding domain for attractants
MCPs are membrane spanning proteins with a binding domain on the periplasmic side and a cytoplasmic domain. The cytoplasmic domain interacts with CheW and CheA The extracellular/periplasmic domain binds specific molecules, well be talking about MCPs that bind attractants such as amino acids and sugars. The MCP proteins are usually grouped in clusters at both ends of the E. coli cell.
CheW Cytoplasmic domain
CheA
Basic signalling to the flagella
CheA is subject to autophosphorylation
CheA CheA
P
CheA hydrolyzes a molecule of ATP to ADP and the phosphate is covalently attached to cheA
ATP
ADP
CheA will then transfer this phosphate to cheY or cheB, cytoplasmic proteins. When cheY is phosphorylated, it diffuses away, eventually interacting with the flagellar motor. When cheY interacts with the motor, it causes the flagella to start spinning clockwise, causing the cell to tumble. After a few seconds, cheZ removes the phosphate from cheY, and the flagella resumes CCW rotation (the cell swims straight again) If cheY is able to cause even one flagella to start spinning CW (remember, E. coli typically has multiple flagella), its enough to cause the cell to tumble.
Flagella switches to CW
P P
P
CheY
P
cheZ
Flagella resumes CCW
Ligand binding to the receptor modifies cheA activity
Amino acids, sugars, or other attractant ligands bind to receptor
Binding of attractant ligands to receptor inhibits the rate of cheA autophosphorylation.
i.e. the attractant ligands inhibit the activity of the receptor
Conformational shift in MCP
Some nomenclature: molecules that bind receptors are called ligands.
ATP hydrolysis and phosphorylation of cheA inhibited X ATP
In contrast, binding of repellants activates cheA
CheW CheA No phosphorylation of cheY, flagella keeps spinning CCW, cell swims straight.
As cheA autophosphorylation decreases, levels of phosphorylated cheY also drop, leading to less frequent tumbles.
cheY
P
cheZ
P
Receptor regulation
adds
cheR methyl groups to MCPs. cheR activity is increased as MCPs bind attractant ligands (attractant binding increase methylase activity) MCPs have multiple (4-6) methylation sites. methyl groups removed The ratio of bound attractants to total methylation level modulates the activity of cheA. by cheB.
If attractant levels decrease, ratio of attractant Inhibited by attractant to methyl groups is briefly lower. This activates binding (through cheA) cheA, causing a tumble (though cheY) and activating cheB. cheB then removes some methyl groups and the bound attractant to CH3 methylation ratio is restored. If attractant levels increase, cheA is inhibited. Therefore, cheB is inhibited methylation levels on the MCP will increase, and the bacteria will make a longer run. the attractant:methylation ratio serves as a memory of what attractant levels were a few second ago. When attractant levels go up, tumbling occurs less frequently until the methylation by cheR catches up When attractant levels go down, the cell activates tumbling until cheB has time to remove methyl groups and restore the ratio.
CH3
P
cheA cheB cheR On rate of methyl groups increases with attractant binding to receptor
Another way to look at it:
Receptor regulation decrease in attractants/increase in cheA autophosphorylase activity repellants ligand binding increases as attractand decreases
cheA then adds a phosphate to either cheY or cheB
Note that there are CheW hundreds~thousands of copies of each molecule inside each cell, so some cheY and cheB can be phosphorylated CheA activated simultaneously by different cheAs.
CH3
P P
When cheB is phosphorylated, it acts as a methylesterase and removes methyl groups from MCPs, increasing their sensitivity Binding of a repellant has an identical effect on the network as a decrease in attractants repellant binding activates cheA, increasing tumbling and activating the demethylase activity of cheB.
cheB
P
CH3
Demethylation of MCP
cheY
Flagella tumbling
Receptor networks
There are multiple chemoreceptors in E. coli. Typically, around two thousand of each kind of receptor are found in a given cell. The receptors are clustered and linked together by cheW and cheA, different kinds of receptors (receptors for repellants or attractors) may interact with the same cheA and cheW proteins. The receptors are able to integrate the combined signal from the local environment, and send this signal to the cheW/cheA complex, controlling tumbling rate of the cell. e.g. a simultaneous increase in both attractants and repellants might cause no change in tumbling frequency Multiple attractants at low concentrations will work better than single attractants (the total amount of attractants and repellants determines the activity of the receptors)
Summing up
Increasing the activation of an MCP causes tumbling
cheA cheY flagella
Following the activation, the sensitivity of the receptor is re-set to a less sensitive level by methylation (or demethylation)
cheA cheB demethylation The increased activity of cheA only lasts until the methylase system adjusts the receptor methylation levels, cheA then returns to its normal basal activity (~95% runs, 5% tumble).
Repellants activate the receptors. Attractants inhibit their activation. .
Ligands Attractants inhibit cheA Repellants activate
cheY MCPs cheW cheA
P P
ADP
P
Tumbling promoted
P
ATP
ADP
P cheB P
cheZ
CH3
CH3
cheR
CH3