-
chemotaxis
- the movement of a cell toward or away from a chemical signal
- - amino acids, sugars, oxygen, electron acceptors
-
swarming assay
- soft agar + compound that attracts the bacteria and can be metabolized
- cells inoculated at center metabolize the compound and swim out in circle up the gradient they have created
- assay only works if bacteria can metabolize the attractant
- takes hours, includes growth
- agarose plug containing attractant is placed in center of liquid suspension of bacteria
- cells accumulate in bright ring surrounding the plug
- does not require growth, response in minutes
-
3-D random walk of chemotaxis
swimming toward greater concentration of attractant - biased towards CCW for longer times
-
Why study chemotaxis?
- mutants have clearly observable phenotypes, but genes are not usually essential
- chemotaxis signaling is incredibly sensitive-cells can respond effectively to very small changes (<1%) in attractant concentration
- chemotaxis signaling is effective over a wide range of attractant concentrations
- - enable bacteria to sense slightly better environment over a wide range of backgrounds
-
relationship between attractant binding and CheA~P
↑ attractant binding → ↓CheA~P
-
basal level of attractant
- basal level of attractant binding to MCP
- basal CheA activity level, making CheA~P
- basal level of CheY~P
- basal level of CheY~P binding to FliM on flagellum
create a baseline level of CheY~P binding to FliM, with flagellar reversal occurring every 2-4 seconds
-
attraction concentration increase
- increased attractant binding to MCP
- decreased CheA activity, making less CheA~P
- less phosphotransfer to CheY, making less CheY~P
- less CheY~P binding to FliM on flagellum
- CheZ deactivates CheY
effect is less CW rotation, fewer tumbles, longer runs
-
attraction concentration decrease
- decreased attractant binding to MCP
- increased CheA activity, making more CheA~P
- more phosphotransfer to CheY, making more CheY~P
- more CheY~P binding to FliM on flagellum
- CheZ deactivates CheY
effect is more CW rotation, more tumbles, shorter runs
-
How does a bacterium know if it is swimming up a gradient of attractant?
- not by spatial sensing
- use a temporal sensing mechanism (more signal now than there was a few seconds ago?)
- to compare now to a few seconds ago, bacteria reset their signaling system rapidly - current attractant concentration they currently see becomes the new norm
-
adaptation
- resetting the signalling baseline to reflect current conditions
- return of CheA activity to pre-stimulus level
-
basal level of attractant adaptation
- basal level of attractant binding to MCP
- basal CheA activity level, making CheA~P
- basal level of CheY~P
- basal level of CheY~P binding to FliM
- CheZ deactivates CheY
- CheR methylates MCP
- CheA phosphorylates CheB
- CheB demethylates MCP
-
attraction concentration increase adaptation
- increased attractant binding to MCP
- decreased CheA activity level, making less CheA~P
- less CheY~P
- less CheY~P binding to FliM
- CheZ deactivates CheY
- CheR methylates MCP
- CheA phophorylates CheB less
- CheB demethylates MCP less
- 1-5 create the initial response
- 6-8 cause buildup of methyl groups on MCP -> increases its ability to stimulate CheA
- causes adaptation
-
attraction concentration decrease adaptation
- decreased attractant binding to MCP
- increased CheA activity level, making more CheA~P
- more CheY~P
- more CheY~P binding to FliM
- CheZ deactivates CheY
- CheR methylates MCP
- CheA phosphorylates CheB more
- CheB demethylates MCP more
- 1-5 create the initial response to stimulus
- 6-8 cause depletion of methyl groups on MCP -> decreases the ability to stimulate CheA
- causes adaption
-
more attractant binding
less CheA~P and less CheY~P
-
more CheY~P binding to FliM
more tumbling
-
more methyl groups on MCP
more ability to stimulate CheA phosphorylation
-
fewer methyl groups on MCP
less ability to stimulate CheA autophosphorylation
-
CheA activity in response to attractant concentration
- the cell shouldn't respond to the absolute level of attractant
- responds to changes in the amount of attractant
- requires adaption to the ambient level
- when attractant level is increased (stimulus)
- rapid decrease in CheA activity (response)
- if cells held at increased concentration, CheA activity returns to pre-stimulus level (adaptation)
reverse is true for rapid decrease in amount of attractant
-
How does the chemotaxis signaling pathway adapt to the current attractant concentration?
CheR constantly adds methyl groups to specific sites on the MCPs -> increases the ability of the MCP to activate CheA at any [attractant]
CheB removes methyl groups from the MCPs -> reducing their ability to activate CheA at any [attractant]. CheB is a response regulator whose receiver domain is phosphorylated by CheA - increases the activity of CheB. CheB is activated at the same time as they CheY pathway
-
evidence that receptors cooperate with each other, rather than working in isolation
the presence of one amino acid can increase the cell's response to a different amino acid
-
trimers of MCPS found in polar caps
- CheW and CheA link the trimers into tight clusters that can influence each other's signalling
- attractant binding by one trimer can sensitize other trimers
|
|