MCP molecules typically have a periplasmic ligand-binding domain for monitoring attractant or repellent levels in the environment and a cytoplasmic signaling domain for communicating with the motor apparatus (1). The MCP signaling domain, highly conserved in structure, forms ternary complexes with two cytoplasmic proteins, CheA, a histidine kinase, and CheW, which couples CheA activity to chemoreceptor control (2, 3). Changes in receptor ligand occupancy trigger conformational changes in the signaling domain that in turn modulate the flux of phosphoryl groups from CheA to effector proteins that elicit the behavioral response (4, 5). MCPs are capable of detecting chemoeffector concentration changes of only a few parts per thousand over more than a five-log concentration range (6-9). The amplification mechanisms responsible for the high-gain signaling characteristics of bacterial chemoreceptors are still poorly understood but may rely on novel signaling principles that will prove to be widely used in biological signal transduction systems.Like many membrane receptors, MCP molecules are not uniformly distributed but rather clustered, typically at the cell poles (10). The CheA and CheW proteins also localize to these receptor clusters and are largely responsible for their integrity (10), suggesting that bacterial chemoreceptors form a two-dimensional lattice that is held together by bridging connections to CheA and CheW. Bray and colleagues (11-13) have theorized that receptors in such an array might, through conformational coupling, communicate their signaling states to neighboring receptors to produce a large gain in detection sensitivity. Experimental work, primarily with Escherichia coli, provides growing support for this notion.E. coli uses five MCP-family receptors to promote chemotactic movements toward different attractant compounds: Tar (aspartate and maltose), Tsr (serine), Tap (dipeptides), Trg (ribose and galactose), and Aer (oxygen and other electron acceptors). Tap, Trg, and, most likely, Aer are present in the cell at roughly 10% the levels of Tsr and Tar (14). Several lines of evidence indicate that high-and low-abundance receptors might signal collaboratively, and that clustering enhances their detection sensitivity. First, the ability of low-abundance receptors to mediate chemotactic responses implies that they are able to exert control over a substantial fraction of the CheA signaling molecules associated with the receptor array. Second, high-abundance receptors assist one another (15) and low-abundance receptors (16-18) in achieving the methylation changes needed to adapt to sensory stimuli. Third, a multivalent galactose ligand that promotes clustering of Trg (19) also served to recruit Tar and Tsr molecules to the cluster and greatly enhanced their detection sensitivity, implying that communication between receptors in a cluster produces signal amplification (20).In vitro studies of receptors and receptor fragments indicate that more than one receptor signaling domain is needed to form a ternar...

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.