Chemotactic signaling studied by FRET

In a model bacterium, Escherichia coli, the chemotaxis pathway is well characterized (Figure 1). Receptors at the cell surface detect changes in the concentrations of attractants and generate shifts in the level of phosphorylation of a diffusible signaling protein CheY, also called the response regulator. Phosphorylated CheY modulates the direction of flagellar motor rotation and

Figure 1: Signal transduction pathway in chemotaxis of E. coli. Pathway is schematically divided in two parts, excitation and adaptation.

thus affects swimming behavior of the cell. The entire signal transduction pathway includes five attractant-specific receptors (Tsr, Tar, Trg, Tap, and Aer), six cytoplasmic chemotaxis proteins (CheA, CheW, CheR, CheB, CheY, and CheZ), and three proteins comprising a switch complex at the cytoplasmic face of the flagellar motor (FliG, FliM, and FliN). Based on genetic and biochemical data, functions have been assigned to all the chemotaxis proteins: CheA is a kinase that phosphorylates the response regulator CheY and also the methylesterase CheB; CheW is an adaptor protein, coupling CheA to receptors; CheR, a methyltransferase, and CheB, a methylesterase, mediate adaptation to a constant attractant concentration by adjusting the methylation level of receptors; CheZ is a phosphatase of CheY~P. Despite its relative simplicity, the chemotaxis system exhibits features common to many cellular networks, such as signal amplification, integration, and adaptation. This has inspired a number of recent attempts to mathematically model this pathway.

Figure 2: Using FRET between CFP and YFP to measure protein interactions. When CheZ-CFP and CheY-YFP fusion proteins do not interact, illumination with cyan (~430 nm) light results in stronger CFP fluorescence (left). As a result of phosphorylation-induced interactions between CheZ and CheY, CFP and YFP are brought into proximity and excitation energy is transferred, resulting in stronger YFP fluorescence (right).

To better understand how the pathway works in vivo, we used a technique called fluorescence resonance energy transfer (FRET). FRET, which depends on the distance-dependent transfer of energy from an excited donor fluorophore to an acceptor fluorophore, is one of the few tools available to study protein interactions in the cell in real time. In vivo-FRET was greatly advanced by the recent (and ongoing) improvement in spectral characteristics of green fluorescent protein (GFP) that led to creation of spectrally shifted variants of GFP, e.g., cyan and yellow fluorescent proteins (CFP and YFP). Genetically fusing CFP and YFP to CheZ and CheY, respectively, allows one to monitor their interactions in vivo (Figure 2).

At steady state, the rate of synthesis of CheY~P, the molecule that couples the receptors to the flagella, is equal to its rate of destruction. The rate of synthesis is proportional to the activity of the receptor kinase, CheA. The rate of destruction is proportional to the concentration of the enzyme substrate complex, CheZ CheY~P, which can be monitored by FRET. So we measure the cyan and yellow fluorescence, add or remove attractant or repellent, and measure the cyan and yellow fluorescence again. From these measurements, we infer the corresponding change in the activity of the receptor kinase.

We found, to our delight, that the fractional change in the activity of the kinase is many times larger than the fractional change in receptor occupancy. That is, there is a large amplification step at the beginning of the sensory transduction pathway. The amplification appears to be due to receptor-receptor interactions. In addition, using FRET between CFP fused to FliM and YFP fused CheY, we studied the binding of CheY~P to FliM, the interaction that promotes clockwise flagellar rotation.

References

Sourjik, V. and Berg, H.C. Receptor sensitivity in bacterial chemotaxis. Proc. Natl. Acad. Sci. USA 99, 123-127 (2002).

Sourjik, V. and Berg, H.C. Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 99, 12669-12674 (2002).

Sourjik, V. and Berg, H.C. Functional interactions between receptors in bacterial chemotaxis. Nature 428, 437-443 (2004).

Chemotactic signaling studied by BRET

Another way to do FRET is to use a luciferase as the fluorescence donor, a technique called bioluminescence resonance energy transfer (BRET). This allows one to study FRET in cells swimming in a cuvette, without having to provide excitation light. We used this technique to ask whether the receptor kinase knows what the flagellar motors are doing. The activity of the kinase was monitored by energy transfer between CheZ-Ronilla luciferase and CheyY-YFP (see Chemotactic signaling studied by FRET), while motors were jammed by addition of anti-filament antibody (which crosslinks adjacent filaments in flagellar bundles). Jamming motors did not perturb receptor kinase activity. Naively, one would think that a feedback loop would be employed to turn up kinase activity whenmotors spin exclusively CW. Assuming that stopping motors would perturb such feedback, this does not appear to be the case.

References

Shimizu, T.S., Delalez, N., Pichler, K. and Berg, H.C. Monitoring bacterial chemotaxis by using bioluminescence resonance energy transfer: absence of feedback from the flagellar motors, Proc. Natl. Acad. Sci. USA 103, 2093-2097 (2005).

Sourjik, V., Vaknin, A., Shimizu, T.S. and Berg, H.C. In vivo measurement by FRET of pathway activity in bacterial chemotaxis. Meth. Enzymol. 423, 365-391 (2007).