No abstract

The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants.The Aer protein of Escherichia coli promotes movement toward optimal concentrations of oxygen and other electron acceptors (8,20). Aer homologs have been found in many other species of bacteria, where they probably mediate similar aerotactic behaviors. Aer communicates with the flagellar motors through a cytoplasmic signaling domain that modulates the activity of the histidine kinase CheA to control the phosphorylation state of the CheY response regulator. The Aer signaling domain is closely related to that of transmembrane chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family, but Aer differs from orthodox MCPs in several important respects. First, the Aer signaling domain does not employ reversible methylation changes for sensory adaptation. Second, the Aer molecule, although membrane associated, does not have a periplasmic sensing domain but rather a cytoplasmic PAS domain that is thought to sense oxygen stimuli by monitoring cellular electron transport activity. PAS domains are widespread in proteins that sense cellular energy levels, redox potential, light, and other stimuli (24). The PAS structural motif comprises a binding pocket that can be adapted for a variety of small-molecule ligands, such as heme (in FixL). In the case of Aer, the PAS ligand is flavin adenine dinucle...