Quantitative assessment of oxygen availability: perceived aerobiosis and its effect on flux distribution in the respiratory chain of Escherichia coli

Abstract

Despite a large number of studies on the role of oxygen in cellular processes, there is no consensus as to how oxygen availability to the cell should be defined, let alone how it should be quantified. Here, a quantitative definition for oxygen availability (perceived aerobiosis) is presented; the definition is based on a calibration with reference to the minimal oxygen supply rate needed for fully oxidative catabolism (i.e., complete conversion of the energy source to CO(2) and water for glucose-limited conditions). This quantitative method is used to show how steady-state electron fluxes through the alternative cytochrome oxidases of Escherichia coli are distributed as a function of the extent of aerobiosis of glucose-limited chemostat cultures. At low oxygen availability the electron flux is mainly via the high-affinity cytochrome bd oxidase, and, at higher oxygen availability, a similar phenomenon occurs but now via the low-affinity cytochrome bo oxidase. The main finding is that the catabolic activities of E. coli (and specifically its respiratory activity) are affected by the actual oxygen availability per unit of biomass rather than by the residual dissolved oxygen concentration of the culture.

FIG. 1.

Effect of oxygen supply on steady-state acetate production rate (q_Acet) in two chemostats. The agitation rate was fixed at 500 rpm. Solid circles, Bioflo 3000 (A); open circles, Bioflo III (B). Lines are linear regressions through the two sets of data points.

FIG. 2.

Effect of agitation rate on steady state acetate production (q_Acet) in a Bioflo 3000 at fixed oxygen supply (air, 20.95% O₂). Open circles, stepped-up agitation rate; solid circles, stepped-down agitation rate.

FIG. 3.

Effect of oxygen supply on the steady-state acetate production rate (q_Acet) in two chemostats. Solid circles, Bioflo 3000; open circles, Applicon type. In the initial phase of each run cells were supplied with air (20.95% O₂). Agitation rate was fixed at the minimal value at which cells produce no acetate (i.e., 568 and 610 rpm for the two chemostats, respectively). This point is defined as 100% aerobiosis. Next oxygen availability was varied by varying oxygen percentage in the input gas.

FIG. 4.

Contribution of cytochrome d (open squares) and cytochrome o (open circles) to total respiratory activity, calculated on the basis of measured cytochrome d content (1), rDOT values (Fig. 5), respiration rates (qO₂, closed squares), and known K_m and V_max values for the cytochrome bd oxidase (14) (see also Materials and Methods).

FIG. 5.

Effect of oxygen availability on microaerobic (below 100% aerobiosis) steady-state rDOT. Bars, amplitudes of the oscillations observed (see Results). Inset, complete range.