Monitoring bacterial chemotaxis by using bioluminescence resonance energy transfer: absence of feedback from the flagellar motors.
We looked for a feedback system in Escherichia coli that might sense the rotational bias of flagellar motors and regulate the activity of the chemotaxis receptor kinase. Our search was based on the assumption that any machinery that senses rotational bias will be perturbed if flagellar rotation stops. We monitored the activity of the kinase in swimming cells by bioluminescence resonance energy transfer (BRET) between Renilla luciferase fused to the phosphatase, CheZ, and yellow fluorescent protein fused to the response regulator, CheY. Then we jammed the flagellar motors by adding an antifilament antibody that crosslinks adjacent filaments in flagellar bundles. At steady state, the rate of phosphorylation of CheY is equal to the rate of dephosphorylation of CheY-P, which is proportional to the degree of association between CheZ and CheY-P, the quantity sensed by BRET. No changes were observed, even upon addition of an amount of antibody that stopped the swimming of >95% of cells within a few seconds. So, the kinase does not appear to be sensitive to motor output. The BRET technique is complementary to one based on FRET, described previously. Its reliability was confirmed by measurements of the response of cells to the addition of attractants.
Shimizu
Thomas S
TS
Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
Delalez
Nicolas
N
Pichler
Klemens
K
Berg
Howard C
HC
D000907:Antibodies, Bacterial / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D002633:Chemotaxis / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D005407:Flagella / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049410:Luciferases, Renilla / Q000032:analysis* / Q000235:genetics; D049449:Luminescence; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D000072236:Methyl-Accepting Chemotaxis Proteins; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011993:Recombinant Fusion Proteins; C000607606:cheY protein, E coli; C054562:yellow fluorescent protein, Bacteria; D049410:Luciferases, Renilla