4.8 Article

Monitoring bacterial chemotaxis by using bioluminescence resonance energy transfer: Absence of feedback from the flagellar motors

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NATL ACAD SCIENCES
DOI: 10.1073/pnas.0510958103

关键词

Escherichia coli; signal transduction; Renilla; luciferase

资金

  1. NIAID NIH HHS [AI063747, AI016478, R01 AI016478, R37 AI016478, F32 AI063747] Funding Source: Medline

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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.

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