Abstract
We used mixed, mutagenic oligonucleotides to create single amino acid substitutions in the bacterial chemoreceptor Trg. Mutagenesis was directed at a 20-residue segment of the periplasmic domain implicated in ligand recognition. Transmembrane signaling by the mutant receptors was assayed in vivo by monitoring adaptational covalent modification. Among 20 functionally altered but stable receptors there were two distinct signaling phenotypes. Insensitive receptors did not signal upon stimulation and thus appeared defective in productive ligand interaction. Mimicked-occupancy receptors exhibited transmembrane signaling without ligand. Many mimicked-occupancy receptors produced additional signaling upon ligand binding and in appropriate conditions mediated effective chemotaxis; most insensitive receptors did not. Like normal receptors with one binding site occupied, mimicked-occupancy proteins adapted to persistent transmembrane signaling by increased methylation and thus could respond to other stimuli. Signaling phenotypes were strikingly segregated by residue position. Substitutions mimicking ligand occupancy occurred in half the segment, and those creating insensitive phenotypes occurred in the other half. These observations could be related to the three-dimensional structure of the periplasmic domain of the Tar(s) chemoreceptor. Insensitive substitutions occurred near the distal end of helix 1, where bulky protein ligands could interact; occupancy-mimicking substitutions were on the same helix at positions buried in the subunit interface between helices 1 and 1'. Thus perturbation of the interface induced transmembrane signaling, implicating changes at that interface in signal transduction, a conclusion consistent with differences in crystal structures of unoccupied and ligand-occupied Tar(s).