The sensor kinase KdpD and the response regulator KdpE control induction of the kdpFABC operon encoding the high-affinity K+-transport system KdpFABC in response to K+ limitation or salt stress. Under K+ limiting conditions the Kdp system restores the intracellular K+ concentration, while in response to salt stress K+ is accumulated far above the normal content. The kinase activity of KdpD is inhibited at high concentrations of K+, so it has been puzzling how the sensor can be activated in response to salt stress. Here, we demonstrate that the universal stress protein UspC acts as a scaffolding protein of the KdpD/KdpE signaling cascade by interacting with a Usp domain in KdpD of the UspA subfamily under salt stress. Escherichia coli encodes three single domain proteins of this subfamily, UspA, UspC, and UspD, whose expression is up-regulated under various stress conditions. Among these proteins only UspC stimulated the in vitro reconstructed signaling cascade (KdpD→KdpE→DNA) resulting in phosphorylation of KdpE at a K+ concentration that would otherwise almost prevent phosphorylation. In agreement, in a ΔuspC mutant KdpFABC production was down-regulated significantly when cells were exposed to salt stress, but unchanged under K+ limitation. Biochemical studies revealed that UspC interacts specifically with the Usp domain in the stimulus perceiving N-terminal domain of KdpD. Furthermore, UspC stabilized the KdpD/KdpE∼P/DNA complex and is therefore believed to act as a scaffolding protein. This study describes the stimulation of a bacterial twocomponent system under distinct stress conditions by a scaffolding protein, and highlights a new role of the universal stress proteins.