Monitoring Two-Component Sensor Kinases with a Chemotaxis Signal Readout

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1729)

Abstract

Bacteria use two-component signal transduction systems to elicit adaptive responses to environmental changes. The simplest of these systems comprises a transmembrane sensor with histidine kinase activity and its cytoplasmic response regulator partner. Stimulus-response studies of two-component signaling systems typically employ expression reporters, such as β-galactosidase, that operate with relatively slow kinetics and low precision. In this chapter, we illustrate a new strategy for directly measuring the signaling activities of two-component sensor kinases in vivo. Our method exploits recent work that defines the recognition determinants for sensor-response regulator signaling transactions, which enabled us to couple histidine kinases to a FRET-based assay that uses signaling components of the E. coli chemotaxis system. We demonstrate the approach with NarX, a nitrate/nitrite sensor kinase, but the method should be applicable to other two-component sensor kinases.

Keywords

Förster resonance energy transfer (FRET) Histidine kinase Response regulator Protein interaction specificity Crosstalk 

Notes

Acknowledgment

Our work is supported by research grant GM19559 from the National Institute of General Medical Sciences. DNA sequencing and primer synthesis were carried out by the Protein-DNA Core Facility at the University of Utah, which receives support from National Cancer Institute grant CA42014 to the Huntsman Cancer Institute.

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of BiologyUniversity of UtahSalt Lake CityUSA

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