Discovery of the signal transducer for aerotaxis in E. coli

Discovery of the signal transducer for aerotaxis in E. coli by Sergei Bibikov and Sandy Parkinson
Aerotaxis, the movement of a cell or organism toward or away from oxygen, was first described in bacteria more than a century ago by Engelmann, Pfeffer and Beijerinck, who observed accumulation of cells near air bubbles or other sources of oxygen (see Fig. 1, top panel). Despite considerable study, the molecular mechanism underlying aerotactic behavior has remained elusive. Does the organism detect oxygen directly or does it instead sense some metabolic consequence of different oxygen environments, for example, fluctuations in its internal energy level?
A gene discovered in the E. coli genome sequencing project appeared to encode a protein that might have aerosensing and flagellar signaling functions (Bibikov et al., 1997). To determine if this open reading frame encoded the long sought aerotaxis transducer for E. coli, we cloned the gene (dubbed aer, for aerotaxis), constructed a large in-frame deletion in its coding region and transferred the gene knockout into the chromosome of an aerotactic strain of E. coli.
Figure 1. The classic air bubble assay for bacterial aerotaxis. Aerotactic cells (E. coli, in this case) in liquid culture are placed under a cover slip on a microscope slide. They quickly consume dissolved oxygen in the medium and accumulate near trapped air bubbles (top panel - The thick black arc on the left is the meniscus of the air bubble.) E. coli cells carrying a knockout mutation in the aer gene, although perfectly motile, fail to accumulate near air bubbles (lower panel). These two cultures had the same overall cell density, indicating that cells throughout the aerotactic culture have migrated to the vicinity of the air bubble.
Several simple behavioral tests revealed that, indeed, a functional aer gene was needed for aerotactic responses, but not for other chemotactic responses. For example, cells of the aer deletion mutant failed to collect around air bubbles (Fig. 1, lower panel). Moreover, the aer mutant spread much more slowly than aerotaxis-competent strains on semi-solid agar plates containing succinate as sole carbon and energy source (Fig. 2).
It seems that the respiratory activity of cells growing on succinate depletes the local oxygen supply and creates an oxygen gradient leading outward from the colony. Aerotactic colonies expand in pursuit of the oxygen remaining in the medium, whereas aerotaxis-defective colonies do not. At high succinate concentrations, the difference in colony size is especially dramatic because aerotaxis-defective mutants remain at the site of inoculation, failing to spread even by random motility (see Fig. 2). This agoraphobic effect evidently arises from the ability of cells growing on succinate to produce and excrete aspartate, a powerful attractant for E. coli, which keeps aerotaxis-defective cells from leaving the colony origin. In contrast, the aerotactic response of wild-type cells enables those on the periphery of the colony to escape the aspartate trap and migrate outward, forming bands of cells congregated at the leading edge of the oxygen gradient (see Fig. 2).
*Figure 2. A newly-devised plate assay for E. coli* aerotaxis.** Cells are inoculated into semi-solid agar containing sodium succinate as the sole carbon and energy source. As they grow and consume the succinate aerobically, they establish an oxygen gradient that leads aerotactic cells away from the colony origin (upper colonies). Aerotaxis-defective cells, although motile, fail to spread. Sections of four plates, with increasing initial concentrations of succinate (left to right), are shown. The distinction between aerotactic and non-aerotactic colonies is most pronounced at the highest succinate level (30 mM).
In a follow-up study, we used the succinate plate method to isolate and characterize a variety of aerotaxis-defective mutants (Bibikov et al., 2000). These and other approaches promise to provide a definitive answer to the long-standing puzzle of how E. coli and other bacteria detect oxygen gradients during aerotaxis. Aer may also prove an excellent prototype for exploring the more general question of how cells evaluate their internal physiological state.
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.