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Abstract:
Most motile bacteria follow spatial gradients of chemical and physical stimuli in their environment. In Escherichia coli and other bacteria, the best characterized chemotaxis is in gradients of amino acids or sugars, but other physiological stimuli such as pH, osmolarity, redox potentials, and temperature are also known to elicit tactic responses. These multiple environmental stimuli are integrated and processed within a highly sophisticated chemotaxis network to generate coordinated chemotaxis behavior, which features high sensitivity, a wide dynamic range, and robustness against variations in background stimulation, protein levels, and temperature. Although early studies relied on behavioral analyses to characterize chemotactic responses in vivo, or on biochemical assays to study the pathway in vitro, we describe here a method to directly measure the intracellular pathway response using Förster resonance energy transfer (FRET). In E. coli, the most commonly used form of the FRET assay relies on the interaction between the phosphorylated response regulator CheY and its phosphatase CheZ to quantify activity of the histidine kinase CheA. We further describe a FRET assay for Bacillus subtilis, which employs CheY and the motor-associated phosphatase FliY as a FRET pair. In particular, we highlight the use of FRET to quantify pathway properties, including signal amplification, dynamic range, and kinetics of adaptation.
