5.2: Motility mutants5.2: Motility mutants kdorfman Thu, 11/10/2011 - 15:24
Mutations and Aberrant Signaling
Media for 5.2Media for 5.2 kdorfman Mon, 11/14/2011 - 17:19
Enough to grow overnight cultures and make dilute early log phase cultures of all 7 chemotaxis strains for use during lab.
Mix tryptone and NaCl in water first.
Bring to final volume.
Divide into volumes suitable for the vessel(s) going into the autoclave. (E.g., 750 mL per 1 Liter bottle.)
Add correct amount of agar (3 g per L) to each container. Leave stir bar in container!
Autoclave 60 min
Sit on stir plate, stirring, until handleable
Pour 25 mL per plate. Plates do not keep well, so pour them the morning of the day they will be inoculated.
4 plates per group. (= 48 minimum) So make 1500 mL. Really need 55 plates + some tiny petri dishes to demonstrate swarm agar consistency.
Inoculate at 7 pm: 13 WT plates + 7 of each of the other strains.
E. coli for 5.2E. coli for 5.2 kdorfman Mon, 11/14/2011 - 18:22
Chemotaxis strainsChemotaxis strains kdorfman Fri, 11/04/2011 - 14:39
- Che A- (run mutant)
- Che B- (tumble)
|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
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.
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.
Growing cells for 5.2Growing cells for 5.2 kdorfman Mon, 11/14/2011 - 18:23
Cultures for swarm plates
Two days before lab:
Note: C (4130) grows poorly. Start it sooner, in a round bottomed centrifuge tube. Spin down and resuspend so there will be enough cells to inoculate the swarm plate.
Shake at 30C
Day before lab:
First thing in the morning
Check cultures and dilute as necessary. The goal is to have dense cultures by 4 pm.
Inoculate 52 swarm plates at 4 pm. Incubate at RT. (Students come in at 9 am, take measurements, put plates in 30C incubator, then come back at lab time.)
13 cultures with WT
7 of each of the 6 different Chemotaxis strains.
Start 3 mL overnight cultures of all 7 strains in Minimal medium + HMLTT. Shake at 30C
OR use the same cultures to stab swarm plates as to make usable cultures for motility studies the next day.
Day of lab:
Inoculate ~100 µL of each strain into ~25 mL medium (~200 into 50 for wildtype)
During morning and lab:
Check cell concentration, diluting as needed. Do not let cells get to stationary phase!