Aerotactic responses inredox sensing ͉ prosthetic group ͉ PAS domain ͉ membrane topology M otile bacteria exhibit many adaptive locomotor behaviors, the best studied of which is chemotaxis in Escherichia coli (see refs. 1-3 for recent reviews). These organisms use transmembrane chemoreceptors, known as methyl-accepting chemotaxis proteins (MCPs), to monitor and respond to changes in their chemical environment as they swim about. MCPs have a periplasmic ligand-binding domain that communicates via membrane-spanning segments with a cytoplasmic signaling domain, which forms stable complexes with the CheA and CheW proteins to transmit sensory information to the flagellar motors. Changes in receptor occupancy modulate conformation of the MCP signaling domain, thereby controlling the CheA histidine kinase, whose protein phosphorylation activity regulates the direction of motor rotation. MCPs are excellent models for exploring the molecular mechanisms of transmembrane signaling and sensory adaptation, but, despite extensive study, there are still significant gaps in our understanding of these important processes.The recently discovered Aer protein is an MCP-like transducer that mediates aerotactic (oxygen-seeking) behavior in E. coli (4, 5). The sequence features of Aer suggest that it has an unorthodox domain organization and membrane topology whose study may shed light on the signaling mechanisms in more conventional MCPs (Fig. 1). The N terminus of Aer resembles a segment of NifL, an O 2 -responsive regulatory protein that employs a bound flavin adenine dinucleotide (FAD) molecule as a redox sensor (6, 7).Initial work on Aer suggested that it also bound FAD (4), presumably for sensing aerotactic stimuli in the form of cellular redox changes (8,9). The N termini of Aer and NifL were subsequently shown to contain a PAS motif (10, 11), which in some proteins is known to comprise a binding pocket for a prosthetic group (reviewed in ref. 12). The putative NifL͞PAS aerosensing domain of Aer is followed by a block of predominantly hydrophobic amino acids that may serve to anchor the protein to the cytoplasmic side of the inner membrane (4). The C terminus of Aer has high similarity to the signaling domains of MCPs (4, 5) and may form ternary complexes with CheA and CheW to control the cell's flagellar motors in response to aerotactic stimuli.Here we report initial biochemical and genetic studies of Aer that examine key predictions of this working model. We show that Aer binds FAD noncovalently and that the N-terminal 290 residues of the protein are sufficient for this activity. Fusion of the FAD-binding portion of Aer to the flagellar signaling domain of Tsr, the serine chemoreceptor, yielded a functional aerotaxis transducer, demonstrating that the FAD-binding portion of Aer is sufficient for detecting aerotactic stimuli. We also describe a colony morphology assay for aerotaxis and its use in isolating non-aerotactic point mutants. We found aerotaxisAbbreviations: FAD, flavin adenine dinucleotide; MCP, methyl-accepting che...

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