Chemotaxis strains

for 2014:

Do

  • Che A- (run mutant)
  • Che B- (tumble)
  • tar-
  • ser-
ID (2013) RP__ Genotype Run Tumble 24 hr swarm comments
RP437 437 WT yes yes outer 72mm, inner 62mm sensitive to both
A 1237 cheR- cheB- yes 10 mm adaptation deficient if motile at all (see below)
B (A) 2361 tar- yes yes outer 67mm, inner 58 mm aspartate blind, tumble a lot
C* 4130 cheB- yes 9mm esterase deficient (like 1273)
D (B) 5700 tsr - yes yes 24mm serine blind
E (C) 8611 tsr- tar- tap- trg- yes 8 mm unable to stimulate kinase
F (D) 9353 cheA- yes no 7 mm no response. kinase inactive

*C grows poorly. Start it early, spin down and resuspend to get numbers up.


Odd # groups: WT, A, D, E

Even # groups: WT, B, C, F

Genes
Receptors

  • Tar: aspartate receptor

  • Tsr: serine receptor

  • Trg: ribose/galactose receptor (minor)

  • Tap: peptide receptor (minor)

  • Aer: aerotaxis receptor (minor)

other pathway components

  • CheA: receptor regulated kinase that phosphorylates CheY and CheB. Stimulates activity by forming the receptor--A complex, inhibits activity when attractants bind to receptor-W-A complex.

  • CheW: adaptor protein required to form CheA-receptor complex

  • CheB: Receptor methylesterase. When phosphorylated, removes methyl groups from receptor.

  • CheR: Methyltransferase. Methylates receptors, which stimulates kinase activity. Receptor methylation rate increases when attractants bind.

  • CheY: Response regulator. When phosphorylated, binds to the motor, and promotes CW rotation (increases tumble frequency)

  • CheZ: phosphatase. Inactivates CheY-P.


RP1237
Receptors have four major site of methylation on 4 specific glutamic acids side chains in the cytoplasmic domain portion of the receptor.

For the sake of simplicity lets call them EEEE for glutamic acids (single letter abbreviation E) at sites 1,2,3 & 4.

The students have correctly determined that the EEEE receptor cannot stimulate the kinase (no tumbles), which is the phenotype of the CheR- mutant (the receptor cannot be methylated).

Also, the CheB- mutant is likely to be EmEmEmEm (where Em indicates methyl glutamate), so it is continuously kinase-active (tumbling all the time).

So why does CheR-CheB- tumble?

When receptors are synthesized on the ribosome, two of the four sites are actually glutamine (Q). Thus, the covalent modification state is QEQE in newly-synthesized receptors (primarily the aspartate and serine receptors). In addition to its demethylating activity (Em --> E), CheB can also convert Q --> E. But, without CheB or CheR (CheR-CheB-) the receptors remain QEQE.

It turns out that Q is more like Em than E. So QEQE receptors stimulate enough kinase activity to make them tumble in absence of an attractant stimulus.

When enough attractant is added (aspartate or serine), the cells become smooth swimming (the kinase is inhibited), yet without the adaptation enzyme (CheR & CheB), the cells should stay stimulated until the attractant is removed.


Movies

A: WT
B: run
C: tumble
D: WT
E: run
F: tumble