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