Molecular Oxygen Controls Nitrate Transport of Escherichia coli Nitrate-respiring Cells*

Escherichia coli cells grown anaerobically in the presence of nitrate reduce the nitrate as a terminal electron acceptor in place of molecular oxygen by an induced respiratory-type electron transferring system residing in the inner membrane structure. When oxygen is introduced to a suspension of nitrate-respiring cells, the oxygen is immediately reduced preferentially and the cellular uptake of nitrate ceases abruptly. In contrast, we found that the cells exhibited no oxygen control on uptake of chlorate, a competitive substrate analogue, indicating operation of an oxygen-sensitive transport system specific to nitrate. This was further evidenced by the fact that chlorate inhibition of reduc- tion of nitrate was brought about only when the transport of both chlorate and nitrate was facilitated by the aid of carrier-type chlorate (or nitrate) ionophore. We demonstrated that oxygen inhibition on reduction of nitrate was abolished within the cells treated by octyl glucoside resulting in a removal of permeability barrier specific to nitrate. We conclude that the transient control by molecular oxygen is primarily due to the inhibition of nitrate transport into the cytoplasmic side. Since nitrate induces the nitrate-respiring system, the repression of the nitrate reductase operon by molecular oxygen is consistently interpreted on the basis of the “inducer exclusion mechanism.” from to nitrate coli nitrate-respir-ing consists membrane-bound com-ponents, and reductase-cyto-chrome b%F complex. spatially

contrast, we found that the cells exhibited no oxygen control on uptake of chlorate, a competitive substrate analogue, indicating operation of an oxygen-sensitive transport system specific to nitrate. This was further evidenced by the fact that chlorate inhibition of reduction of nitrate was brought about only when the transport of both chlorate and nitrate was facilitated by the aid of carrier-type chlorate (or nitrate) ionophore. We demonstrated that oxygen inhibition on reduction of nitrate was abolished within the cells treated by octyl glucoside resulting in a removal of permeability barrier specific to nitrate. We conclude that the transient control by molecular oxygen is primarily due to the inhibition of nitrate transport into the cytoplasmic side. Since nitrate induces the nitrate-respiring system, the repression of the nitrate reductase operon by molecular oxygen is consistently interpreted on the basis of the "inducer exclusion mechanism." The respiratory electron transfer from formate to nitrate in Escherichia coli grown anaerobically under nitrate-respiring conditions consists of two major membrane-bound components, formate dehydrogenase and nitrate reductase-cytochrome b%F complex. The complex has been shown to be spatially assembled within the inner membrane from cytochrome bg:; moiety with the electron-accepting site toward the periplasmic side, and nitrate reductase moiety with the nitrate-interacting site toward the cytoplasmic side (1). Under such a topological situation, nitrate, which should permeate the inner membrane to interact with nitrate reductase, is possibly subjected to a transmembrane control.
The inhibitory effect of oxygen, on the other hand, has been known not only on electron transfer to the respiratory nitrate reductase but on the induced synthesis of the enzyme (2,3). However, the role which molecular oxygen plays either in the transient control of the electron transfer or in the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$. To whom correspondence should be addressed.
repression of the induced synthesis so far remains to be elucidated. This communication presents the evidence supporting transient suppression of nitrate transport by molecular oxygen, thus providing a new clue to reveal the novel role played by molecular oxygen.

EXPERIMENTAL PROCEDURES
E. coli (IF0 12433) was cultured anaerobically in a complex medium containing 1% potassium nitrate and 1% sodium formate at 30 "C. Cells were harvested by centrifugation at the log phase and washed three times with 20 mM Tris/HCl containing 200 mM sucrose (pH 8.0) without any anaerobic treatment. The two-electrodes technique to simultaneously monitor oxygen and nitrate (or chlorate) levels in the medium of the cell suspension was essentially the same as previously described (3). The nitrate reductase assay with bipyridylium radical was as previously described (4).

RESULTS AND DISCUSSION
Fig. l a shows that nitrate respiration of intact cells ceases abruptly after the introduction of oxygen, but immediately recommences upon the removal, in agreement with previous observations (2, 3). In response to the cellular uptake of nitrate as monitored by nitrate electrode, the simultaneous appearance of approximately stoichiometric levels of nitrite was confirmed with the whole broth in the presence and absence of oxygen, indicating the transport process as the apparently rate-limiting step. Uptake of chlorate, added as a substrate analogue for nitrate reductase in the absence of oxygen, proceeded, but at a rate as low as that of nitrate uptake under oxygen control, as shown in Figs. l b and 3b, in accordance with a previous report (5). Essentially no oxygen control of chlorate uptake under the same conditions as for nitrate was found (Fig. lb). When nitrate was added to such a cell suspension, the oxygen control occurred as shown in Fig. l b , also confirming a previous observation with Puracoccus denitrijicuns (3). The results indicate that E. coli can thus distinguish nitrate from chlorate in the absence of oxygen, resulting in preferential reduction of nitrate, but not in the presence of oxygen, probably because of a permeability barrier specific to nitrate. Since it has been concluded that the nitrate-interacting site of nitrate reductase is located on the cytoplasmic side of the inner membrane (5)(6)(7)(8), nitrate or even chlorate should be able to permeate the membrane to interact with nitrate reductase. The restricted reduction rate of nitrate in the presence of oxygen and that of chlorate in its absence are assumed to be limited by a transmembrane diffusion process. On the contrary, the reduction of nitrate, stimulated in the absence of oxygen, may be controlled by an oxygensensitive nitrate transport system functioning prior to the onset of the electron transfer toward nitrate.
The above possibility is supported by the following observations (Fig. 2). The reduction rate of nitrate in intact cells assayed with the use of dithionite-reduced methyl viologen or the diquat radical as an electron donor, both impermeable to the inner membrane (7-9) and transferring the electron from the periplasmic side via cytochrome bB;, is hardly affected by the presence of chlorate ions, as expected from their impermeability. On the other hand, when benzyl or heptyl viologen radicals (reported to be a nitrate (or chlorate) ionophore of the carrier type, thus promoting the nitrate (or chlorate) transport (9) The nitrite produced was determined as previously described (4). The rate of nitrite disappearance was confirmed to be negligible under the present conditions. increase in chlorate concentration decreases the rate of nitrate reduction (Fig. 2). This result indicates that nitrate and chlorate are both transported to the cytoplasmic side by the aid of the carrier-type ionophore. Furthermore, the rate of nitrate reduction with benzyl viologen or heptyl viologen higher than that with methyl viologen or diquat implies that ionophore-aided nitrate transport into the cells increases the rate of nitrate reduction, further indicating that nitrate transport is the rate-limiting step in the overall process of nitrate reduction.
If oxygen inhibits nitrate transport as above postulated, then it would be expected that the removal of the permeability barrier to nitrate would abolish the oxygen control of nitrate respiration. In fact, a t half of the critical micelle concentration of octyl glucoside showing to abolish the permeability barrier to chlorate (Fig. 3a), the uptake of chlorate becomes much higher, but still without oxygen control. Octyl glucoside could be incorporated into the membrane bilayer structure, thus increasing the permeability of both anions without critical impairment of the membrane assembly (lo), as is the case of . I , . , . To the medium, were added 0.15 M potassium nitrate, 1.0 ml of cell suspension (4.7 mg of protein), 3.1% hydrogen peroxide, and 15% octyl glucoside (final concentration 0.45%), as indicated in b. The reaction conditions were otherwise essentially the same as in Fig. 1. OG, octyl glucoside.

Time
Triton X-100 (3). It was discovered that, at the octyl glucoside level where the permeability barrier to chlorate is shown to be abolished, the oxygen inhibition of nitrate reduction dramatically disappears and nitrate reduction resumes at an appreciable rate even in the presence of oxygen (Fig. 36). From these results, it is concluded that oxygen primarily inhibits nitrate transport but not the subsequent electron transfer to nitrate.
Jones et al. (8) proposed a possible mechanism of nitrate transport by a nitrate/nitrite antiport either by itself or by the coupling of protonfnitrate and protonfnitrite symports. Oxygen may interact a t a specific site in the nitrate transport system directly or, if coupled, in a proton transport system indirectly. Since the oxygen control was scarcely operative in cells treated with N-ethylmaleimide (10 mM at 30 "C for 30 min) (data not shown), the transport site may involve redoxsensitive sulfhydryl groups undergoing reversible oxidation to the disulfide form in the presence of oxygen (11). The detailed molecular mechanism of how oxygen inhibits nitrate transport remains to be further explored.
It is known that the inducer of the lactose operon (e.g. lactose) is excluded when glucose enters the cell (12). Our results support such an inducer exclusion mechanism in which the oxygen control of nitrate respiration is analogous to the glucose inhibition of lactose transport. Oxygen not only transiently inhibits nitrate respiration, but also represses the

Oxygen Control
of Nitrate Transport 9443 induction of the system by nitrate by inhibiting specifically the nitrate transport system. After submission of the present paper, we realized that Hernandez and Rowe (13) reported that inhibition of nitrate utilization by oxygen appeared to be at the level of nitrate uptake in denitrifying Pseudomonas aeruginosa. It is likely that a common mechanism exists for oxygen control of nitrate uptake in nitrate-respiring microorganisms.