Cooperativity in Signal Transfer through the Uhp System ofEscherichia coli

Abstract

The UhpABC regulatory system in enterobacteria controls the expression of the hexose phosphate transporter UhpT. Signaling is initiated through sensing of extracellular glucose 6-phosphate by membrane-bound UhpC, which in turn modulates the histidine-protein kinase UhpB. Together with the cytoplasmic response regulator UhpA, they constitute a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Activated (i.e., phosphorylated) UhpA binds to the promoter region of uhpT, resulting in initiation of transcription. We have investigated the contribution of transmembrane signaling (through UhpBC) and intracellular activation (through UhpA) to the overall Uhp response (UhpT expression) in vivo. UhpA activation could be made independent of transmembrane signaling when ⌬uhpBC cells were grown on pyruvate. Bacteria must be able to accurately respond to a large number of extracellular signals in their continuously changing surroundings. Therefore, they have evolved sophisticated sensory mechanisms coupled to intracellular signal transduction pathways. The majority of these prokaryotic intracellular signaling routes are based on the reversible phosphorylation of sensor kinases and response regulators in so-called two-component regulatory systems (23,28,30). According to this concept, sensory input affects autophosphorylation on a conserved histidine residue in the transmitter module of the kinase. The phosphoryl group is subsequently transferred to a conserved aspartate residue in the (amino-terminal) receiver domain of the cognate response regulator. This covalent modification elicits a molecular switch, and above a certain threshold amount of phosphorylated regulator, a specific response is turned on, which is in most cases gene transcription via (enhanced) affinity of the carboxyl-terminal output domain of the regulator for the target promoter sequence.To provoke a cellular response, the environmental physical or chemical stimulus has to cross the membrane. Therefore, in most cases the first step in a signal transduction cascade is the activation of a membrane-bound protein. In order to be able to quantitatively study specific transmembrane (receptor) signaling leading to cellular activation, the first (ligand-induced) step and the subsequent response should be well defined and straightforward to quantify. However, despite the huge number of characterized signaling pathways, such examples are not abundant.An exception is the Uhp regulatory system in Escherichia coli (for a review, see reference 13). This signaling pathway is triggered by external glucose 6-phosphate, which is recognized by the membrane-bound receptor protein UhpC. UhpC interacts with a second membrane-bound protein, UhpB, the kinase/phosphatase of the Uhp two-component system. Sensing of glucose 6-phosphate is supposed to cause a conformational change in the supposed UhpBC complex, leading to histidine autophosphorylation in the (cytoplasmic) transmitter domain of UhpB. Upon phosphoryl transfer, the response regulator UhpA (pre...

Application of comparative genomics in the identification and analysis of novel families of membrane-associated receptors in bacteria

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