Functional architecture of an aerotaxis signal transducer

Functional architecture of an aerotaxis signal transducer by Sergei Bibikov and Sandy Parkinson
The aer gene of E. coli mediates aerotactic behavior - movement toward the oxygen concentration best-suited to an organism's metabolic lifestyle. To determine how the Aer transducer senses environmental oxygen gradients and controls flagellar movements, we used a variety of molecular and genetic tools to analyze its strucure-function organization.
Some proteins use prosthetic groups, such as heme, for aerosensing. High level expression of the Aer protein, which copurified with the cellular membrane fraction (Fig. 1), produced membranes with a distinct greenish yellow color, implying that Aer might contain a chromophore. Conceivably, the Aer protein sequestered the chromophore, causing the cells to compensate by increasing its production. The colored substance in Aer-containing membranes proved to be noncovalently associated with the Aer molecule and was subsequently identified by mass spectrometry and other analytical methods as flavin adenine dinucleotide (FAD).
Figure 1. High level expression and membrane location of the Aer protein. Cells carrying an inducible Aer expression plasmid (+) or a control plasmid (-) were collected by centrifugation and disrupted in a French press. Membranes were pelleted by ultracentrifugation and samples of the unfractionated cell lysates, the membrane pellets and the cytoplasmic supernatants were subjected to electrophoresis on a 10% polyacrylamide SDS gel. Proteins were visualized by Coomassie staining, with the positions of various size markers indicated on the left. OmpC and OmpF are prominent outer membrane proteins typically present in the membrane fraction.
The region of the Aer molecule responsible for FAD-binding was identified by isolating and testing a random collection of aerotaxis-defective aer mutants. All mutations in the N-terminal half of Aer drastically reduced its FAD-binding ability, whereas those in the C-terminal half did not. Polypeptide fragments spanning the N-terminal half of the Aer protein were able to bind FAD, confirming that this part of the molecule is both necessary and sufficient for FAD-binding. When the FAD-binding portion of Aer was spliced to the signaling domain of the serine chemoreceptor, the resultant hybrid transducer mediated aerotactic behavior, demonstrating that the FAD-binding portion of Aer is responsible for aerosensing.
To determine whether the central hydrophobic segment of Aer (see Fig. 2) was important for membrane association, we replaced the hydrophobic residues with short, alanine-rich linkers. The deleted proteins failed to associate with the cytoplasmic membrane and did not support aerotactic ability. High level expression of the proteins did not cause a concomitant elevation in the cellular content of FAD, suggesting that membrane insertion might play a role in FAD binding. These findings indicate that Aer uses FAD as a prosthetic group to sense aerotactic stimuli (Fig. 2). Aer most likely associates with the cytoplasmic membrane through its central hydrophobic segment, which is just long enough to traverse the membrane twice. According to this model, both ends of the Aer molecule would be located in the cytoplasm. The C-terminal domain is highly similar to the signaling domains of the MCP family of chemoreceptors, which associate with the cytoplasmic CheW and CheA proteins to generate signals that control the flagellar motors. Aer appears to have a similar signaling capability. The N-terminal portion of Aer binds FAD and functions as an input domain for detecting changes in oxygen environments, perhaps in the form of a redox change in a component of the electron transport chain. Our current studies of Aer aim to test various predictions of this working model with the eventual goal of elucidating how information about the cell's oxygen environment is detected and then conveyed to the Aer signaling domain to elicit an adaptive locomotor response.
Figure 2. Working model of Aer. Most of the molecule is cytoplasmic, but anchored to the inner face of the cytoplasmic membrane by a central segment of hydrophobic amino acids. The N-terminal PAS domain probably binds the FAD prosthetic group, but mutations in the F1 or F2 segment can also disrupt FAD binding. The C-terminal signaling domain interacts with the CheW and CheA proteins of the chemotaxis pathway to transmit control signals to the flagellar motors.
Bibikov, S. I., R. Biran, K. E. Rudd, & J. S. Parkinson (1997) A signal transducer for aerotaxis in Escherichia coli. J. Bacteriology 179:4075-4079.
Bibikov S.I., L.A. Barnes, Y. Gitin, & J.S. Parkinson (2000). Domain organization and FAD-binding determinants in the aerotaxis signal transducer Aer of Escherichia coli. Proc. Natl. Acad. Sci. USA 97:5830-5835.