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In Receptor sensitivity in bacterial chemotaxis by Victor Sourjik and others, Victor Sourjik and Howard C. Berg used a cyan fluorescent tag on CheZ as FRET donor and a yellow tag on CheY as FRET acceptor. Escherichia coli biological cells adhered to a polylysine-coated coverslip in a continuously perfused chamber. A fluorescence microscope excited them with a $442$-nanometre laser; split emission bands fed two photon-counting detectors. The yellow/cyan emission ratio fell when attractant was added and rose when it was removed.^[ref]

Saturating attractant increased cyan emission by an amount matching selective bleaching of the yellow tag: removing the FRET acceptor supported relief of FRET donor transfer. Strains carrying the tagged pair but lacking other chemotaxis machinery showed no stimulus-dependent emission ratio change. Weak illumination reduced bleaching drift.^[ref]

The inference is a change in the population of nearby tagged CheY/CheZ pairs associated with CheY phosphorylation. It neither measures each pair's separation nor directly observes motor motion. The readout also averages many biological cells, and perfusion takes finite time. The pathway interpretation belongs to the chemotaxis FRET assay; the general measurement lesson is that molecular association can alter FRET while the geometry of an individual associated pair stays the same.

Negative feedback closes a causal loop whose net effect opposes an initiating change. In signal transduction, activation followed by a downstream inhibition of the earlier activating stage is one example of negative feedback. Loop sign alone does not establish asymptotic stability or complete signaling adaptation; see the worked signaling feedback example.

A useful biological example is chemotaxis feedback regulation: an Escherichia coli biological cell adjusts its response to a sustained attractant instead of remaining at its immediate response. Increased CheA activity promotes CheB phosphorylation; CheB reverses receptor methylation, opposing their activation. After attractant addition, reduced activity instead favors receptor methylation by CheR, restoring activity. This is a return from receptor activity to the machinery regulating that activity. The response need not return exactly at every dose. ^[ref]

A motivating measurement is the chemotaxis FRET assay. CheY and CheZ carrying yellow and cyan fluorescent tags were observed in immobilized Escherichia coli biological cells on a coverslip in a flow chamber. Adding and removing attractant changed the yellow-to-cyan emission ratio, reporting the association of phosphorylated CheY with CheZ. This measures a molecular readout upstream of motor behavior. Comparisons with biological cells lacking CheR, CheB, or both tested the dependence on receptor modification; these perturbations also change starting activity and sensitivity, so they are not pure changes of one feedback gain. ^[ref]

The later chemotaxis feedback regulation experiment used only the Tar receptor and nonmetabolizable methylaspartate, supplied and removed by flow. Long recordings separated slow recovery from incomplete signaling adaptation; correction for bleaching addressed drift in the optical readout. Activity recovered incompletely after sufficiently large additions, with detectable imprecision already at $10\mu \mathrm{M}$ in that preparation. Restricting the receptor type and preventing consumption of the stimulus help interpret the perturbation, but limit transfer to other receptors and conditions. The readout infers CheA activity through CheY-CheZ association, rather than directly measuring every molecular step. ^[ref]

Negative feedback can also overshoot or sustain oscillations if its return arrives too late. The worked signaling feedback example gives the contrasting instantaneous case; the delayed negative feedback model below isolates the effect of timing. Loop sign describes causation, while asymptotic stability describes what happens to displacements over time.