Differentiation between electron transport sensing and proton motive force sensing by the Aer and Tsr receptors for aerotaxis

Abstract

Aerotaxis (oxygen-seeking) behaviour in Escherichia coli is a response to changes in the electron transport system and not oxygen per se. Because changes in proton motive force (PMF) are coupled to respiratory electron transport, it is difficult to differentiate between PMF, electron transport or redox, all primary candidates for the signal sensed by the aerotaxis receptors, Aer and Tsr. We constructed electron transport mutants that produced different respiratory H+/e- stoichiometries. These strains expressed binary combinations of one NADH dehydrogenase and one quinol oxidase. We then introduced either an aer or tsr mutation into each mutant to create two sets of electron transport mutants. In vivo H+/e- ratios for strains grown in glycerol medium ranged from 1.46+/-0.18-3.04+/-0.47, but rates of respiration and growth were similar. The PMF jump in response to oxygen was proportional to the H+/e- ratio in each set of mutants (r2=0.986-0.996). The length of Tsr-mediated aerotaxis responses increased with the PMF jump (r2=0.988), but Aer-mediated responses did not correlate with either PMF changes (r2=0.297) or the rate of electron transport (r2=0.066). Aer-mediated responses were linked to NADH dehydrogenase I, although there was no absolute requirement. The data indicate that Tsr responds to changes in PMF, but strong Aer responses to oxygen are associated with redox changes in NADH dehydrogenase I.

Fig. 1

Scheme for aerobic electron transport system in E. coli, showing H⁺/e^- ratio for individual complexes. Genes encoding each complex are shown in parentheses.

Fig. 2

Determination of H⁺/e^- ratio in wild-type (RP437) E. coli by measuring the acidification of the medium following injection of air-saturated water. Bacteria were grown in glycerol medium as described in Experimental Procedures, washed and resuspended in reaction buffer contained 50 mM KSCN and 10 mM glycerol. The extracellular H⁺ concentration (mV) was recorded with an H⁺-sensitive, ion-selective electrode. (A) Cells were sparged with argon gas for a minimum of 10 min to assure anaerobiosis and a stable baseline. (B) The pH was increased to 6.5 by addition of 40 μl of argon-sparged 20 mM KOH. (C) 100 μl air-saturated water (25 nmol O₂) was injected into the incubation chamber. (D) The pH was increased to approximately 6.5 by addition of KOH. (E) The electrode was calibrated with 10 μl (50 nmol H⁺) aliquots of argon-flushed 5 mM HCl.

Fig. 3

Plots comparing the step increase in Δψ (Δψ_O2 - Δψ_N2) versus the H⁺/e^- ratio (upper panel), and the aerotaxis response versus (Δψ_O2 - Δψ_N2) (lower panel) for Aer+ and Tsr+ strains. The data used for these correlation analyses are from Table 1. Each datum point represents the mean value obtained for one electron transport mutant. For Aer⁺ strains, the response times above the best-fit line are from strains expressing NADH dehydrogenase I and those below the line are from strains expressing NADH dehydrogenase II.

Fig. 4

Colony morphology on semi-solid agar with the indicated carbon source. The agar was inoculated and incubated at 30°C. Colonies on tryptone were incubated for 12 h and are shown at 0.25X the magnification of the other colonies, which were incubated for 18 h (designated by underlined text) or 28 h (normal text), depending on the swarm rates. Colonies were photographed digitally with dark-field illumination (experimental procedures). The relevant proteins expressed are shown except where tsr, aer or aer tsr is the only mutation and the wild-type electron transport system is present.

Fig. 5

Capillary assays for aerotaxis. The strains were grown in LB broth to OD_600nm of 0.5 to 0.6 and loaded into glass capillaries as described in the text. The air-liquid interface (meniscus) is visible at the right in each capillary. After incubation for 15 min to 30 min, images were captured using a video microscope (62.5x magnification) with dark-field illumination.

Fig. 6

Model of aerotaxis pathways incorporating the findings of this study. Aer is proposed to sense redox changes, most importantly in NADH dehydrogenase I. Tsr senses changes in PMF. Abbreviations: NDH-1, NADH dehydrogenase I; NDH-2, NADH dehydrogenase II; Cyt bd, cytochrome bd oxidase; Cyt bo, cytochrome bo oxidase; A, CheA histidine kinase; W, CheW docking protein; Y, CheY response regulator; Z, CheZ protein; IM, inner membrane; OM, outer membrane; CW clockwise.