INTRODUCTION

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Escherichia coli can respire anaerobically by using a number of alternative terminal electron acceptors, such as nitrate, nitrite, dimethyl sulfoxide, trimethylamine-N-oxide, and fumarate, in order to generate energy by electron transport-linked phosphorylation reactions (11, 12). To accomplish nitrate reduction, E. coli can synthesize three distinct nitrate reductase enzymes. The napFDAGHBC operon, located at 46.5 min on the chromosome and previously named aeg-46.5 (6), encodes a periplasmic nitrate reductase enzyme homologous to the NapAB enzyme of Alcaligenes eutrophus (Ralstonia eutropha), Rhodobacter capsulatus, and Thiosphaera pantotropha (Paracoccus denitrificans) (1, 3, 15, 19, 20). The narGHJI operon, which has been the subject of intensive study for many years, encodes the major respiratory nitrate reductase located in the cytoplasmic membrane (11, 26). A third nitrate reductase, encoded by the narZYWV operon, is biochemically similar to the NarGHJI enzyme but is constitutively expressed at relatively low levels in the cell (2). The physiological rationale for why E. coli possesses three nitrate reductase operons and when two of these are expressed during anaerobic cell growth is not clear.

In this study we examined the patterns of napF and narG gene expression in response to different steady-state levels of nitrate by using napF-lacZ and narG-lacZ reporter fusions in anaerobic chemostat culture. The product of nitrate reduction, nitrite, was also tested for its ability to alter napF and narG expression. Finally, the levels of nitrate and nitrite in the chemostat vessel were measured to better understand the relationship between nitrate and nitrite consumption and napF and narG expression. The results of these continuous cell culture experiments reveal a differential and complementary pattern of nitrate reductase gene expression whereby the napF operon is expressed preferentially before the narG operon under limiting nitrate conditions.

	MATERIALS AND METHODS

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Bacteria, plasmids, and phages. All experiments were performed with E. coli MC4100 [F (argF-lac)U169 araD139 (argF-lac)U169 rpsL150 deoC1 relA1 flbB5301 rbsR ptsF25] (21). The lacZ reporter fusions used to monitor expression of the two nitrate reductase operons were HW2 (napF-lacZ) (this study) and PC50 (narG-lacZ) (8). Since the respective fusions are integrated at the ^att site on the chromosome, each strain is wild type for the narGHJI and napF operons. The napF-lacZ fusion was constructed by the generation of a DNA fragment containing the napF (aeg-46.5) control region from position 224 to position +169 relative to the start of transcription by using standard PCR protocols (17). The resulting DNA fragment was then inserted into plasmid pRS415 (22) to give the napF-lacZ operon fusion plasmid designated pHW2. The entire DNA insert in pHW2 was DNA sequenced to confirm the intended construction (18). The napF-lacZ fusion was then transferred onto RS45 to generate HW2. A high-titer lysate was then used to introduce the phage into MC4100 as previously described (22).

Cell growth. For routine cell growth and plasmid construction, cells were grown in Luria-Bertani liquid or solid medium. For batch cell culture, cells were grown in a glucose (40 mM) minimal medium (9). Where indicated, sodium nitrate (40 mM) or sodium nitrite (5 mM) was added to the growth medium after sterilization. Anaerobic growth was performed at 37°C in 10-ml anaerobic culture tubes fitted with butyl rubber stoppers (13). Cells grown overnight under identical conditions in the same medium were used for inoculation.

Enzyme assays. -Galactosidase assays were performed as described previously (9). One unit of -galactosidase is defined as the hydrolysis of 1 nmol of o-nitrophenyl--D-galactopyranoside (ONPG) per min per mg of protein. Nitrate reductase activity was measured as previously described (14).

Nitrate and nitrite determination. To determine the concentrations of nitrate and nitrite in the culture medium, high-pressure liquid chromatography (HPLC) methods were used. The analytical conditions were as follows. A Waters 625 LC pump and 600E controller unit were used as the delivery system under ambient temperature conditions; the column was a Whatman Partisphere SAX cartridge (4.6 by 250 mm). A Shimadzu SPD-6AV UV-visible detector was used at a wavelength of 210 nm. The buffer was 50 mM phosphate (pH 3.0), and the elution rate was 1.0 ml/min. The sample injection volume was 50 µl. The sensitivity for nitrate detection was 0.03 µM, and that for nitrite was 0.04 µM.

Materials. ONPG was purchased from Sigma Chemical Co., St. Louis, Mo. X-Gal was obtained from International Biotechnologies, Inc., New Haven, Conn. Casamino Acids was from Difco Laboratories, Detroit, Mich. Nitrogen gas was supplied by Arco, Inc. All other chemicals used in this study were of reagent grade.

	RESULTS AND DISCUSSION

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Effect of nitrate concentration on narG-lacZ expression. To determine how narGHJI operon expression varies in response to different steady-state levels of nitrate, anaerobic continuous cell culture methods were employed with a strain containing a narG-lacZ reporter fusion (Fig. 1A). Gene expression was lowest in cells grown anaerobically in the absence of any added nitrate. When the concentration of nitrate in the added medium was 1 mM, expression was increased by twofold. Maximal narG-lacZ gene expression (i.e., a 90-fold increase) was not seen until the nitrate addition level was 8 mM. The cell has the potential to fine-tune narG gene expression over a wide range in response to nitrate availability. As the nitrate level was increased above 8 mM to 40 mM, gene expression remained relatively unchanged (Fig. 1A and data not shown). Interestingly, at between 3 and 4 mM nitrate added, a modest plateau in narG-lacZ gene expression was seen. This pattern was consistently reproduced in independent experiments (data not shown). It should be noted that since nitrate was continually being consumed by the cells in the chemostat growth vessel, the nitrate addition values shown in Fig. 1A are not the same as the actual level of nitrate present in the growth vessel and, thus, the level needed to induce narG-lacZ expression (see below).

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Effect of nitrate on narG-lacZ and napF-lacZ expression during anaerobic cell growth. The amount of nitrate added via the medium addition to the chemostat vessel is indicated. When cells were shifted to a new condition, steady state was generally achieved within five residence times (see Materials and Methods). (A) Expression of the narG-lacZ fusion and the napF-lacZ fusion. The maximal levels of narG-lacZ and napF-lacZ expression were 16,000 and 5,800 U, respectively. (B) Effect of nitrate concentration on nitrate reductase enzyme activity in the narG-lacZ reporter strain. The chemostat was sampled after each steady state was achieved, and nitrate reductase activity was determined. The fermentor conditions were identical to those used for panel A.

Effect of nitrate concentration on napF-lacZ gene expression. The pattern of napF-lacZ expression seen in response to nitrate additions was strikingly different than that seen for narG-lacZ expression (Fig. 1A). Half-maximal napF gene expression was seen at 0.5 mM nitrate added, the condition where narG-lacZ expression was still minimal. Maximal napF-lacZ expression (ca. 30-fold induction) was achieved at 1 mM nitrate added. At higher levels of nitrate addition, napF-lacZ expression then declined to a near-basal level seen when no nitrate was added (Fig. 1A). At 4 mM nitrate added, napF-lacZ expression was less than 20% of the maximal expression level, conditions where narG-lacZ expression was not yet one-half of its fully induced level. These two nitrate reductase operons are differentially expressed in a complementary fashion in response to changing levels of nitrate addition. The napF operon is induced only within a low substrate concentration range, while the narGHJI operon is induced maximally only when considerably higher substrate levels are achieved. In the nitrate addition range of between 1 and 3 mM, both the NapF and NarG nitrate reductases appear to function simultaneously to consume nitrate, as evidenced by their overlapping patterns of gene expression.

Concentrations of nitrate and nitrite remaining in the culture medium. As noted above, the steady-state level of nitrate remaining in the chemostat vessel must be lower than the concentration of nitrate being added, since the cells are continually removing nitrate by reducing it to nitrite. HPLC methods were employed to measure the levels of nitrate and nitrite present in the vessel (Fig. 2). At nitrate addition levels of below 5.0 mM, no nitrate was detected in the vessel (where the detection limit was less than 0.03 µM nitrate [see Materials and Methods]). Above this addition level, the concentration of nitrate in the vessel was gradually increased in a linear fashion to 4.5 mM as the nitrate additions were increased to 12.5 mM. Within this range, nitrate was clearly present in excess of the cell's capacity to accumulate and reduce it.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Levels of nitrate remaining and nitrite accumulated in the chemostat vessel with different concentrations of nitrate added. Upon the shift to each new condition, steady state was generally achieved within five residence times. The vessel was then sampled, and the concentrations of nitrate and nitrite remaining in the growth medium were determined by HPLC (see Materials and Methods).

Effect of nitrite addition on napF-lacZ and narG-lacZ expression. To establish how napF-lacZ and narG-lacZ expression responds to addition of nitrite, the product of nitrate reduction by nitrate reductase, similar steady-state chemostat experiments were performed where nitrite was added in place of nitrate (Fig. 3). Three observations are readily apparent. First, nitrite additions did not lead to the maximal level of napF-lacZ expression seen when nitrate was added. Rather, nitrite caused a maximal level of induction that was about 25% of that seen for nitrate (i.e., 1,400 versus 5,800 U). To reach these respective maximum levels, a fourfold higher concentration of nitrite addition relative to nitrate addition was needed. For the maximal nitrite induction, the nitrite level remaining in the vessel was between 2 and 4 mM. As noted above, to give maximal gene expression with nitrate, the detectable level of nitrate in the vessel was below 0.03 µM. These findings suggest that nitrate is a stronger regulatory signal than nitrite by at least 2 to 3 orders of magnitude. Finally, napF-lacZ expression remained elevated when nitrite was present (Fig. 3). In contrast, when high levels of nitrate were present, napF-lacZ expression was reduced nearly to the basal level seen when no nitrate or nitrite was present (Fig. 1A). Nitrate, but not nitrite, is therefore required to repress napF-lacZ expression.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Effect of nitrite on narG-lacZ and napF-lacZ expression during anaerobic cell growth. The amount of nitrite present in the medium addition was varied as described for Fig. 1A.

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Level of nitrite remaining in the chemostat vessel with different concentrations of nitrite added. Following the sampling of the vessel as described for Fig. 1B, the nitrite concentration was determined by HPLC (see Materials and Methods).

Does nitrite antagonize napF-lacZ or narG-lacZ expression? To establish whether the cell can effectively discriminate between nitrate and nitrite as a signal for napF-lacZ expression, we examined the effect of nitrite-dependent induction in the presence of 0.5 or 1.0 mM nitrate (Fig. 5). Under these conditions, napF-lacZ expression was near maximal due to the added nitrate. When both anions were added to the vessel simultaneously, the nitrite addition at either an equimolar concentration or an eightfold molar excess relative to nitrate had little to no effect on napF-lacZ expression (Fig. 5A). Similar results were seen when nitrate was present at 1 mM (Fig. 5B). Therefore, nitrite is not a significant coinducer, corepressor, or antagonist of the cells' ability to recognize nitrate as a signal.

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Effect of various nitrite additions on napF-lacZ expression in the presence of 0.5 mM nitrate (A) and 1 mM nitrate (B). The medium addition contained a fixed amount of nitrate and the indicated amounts of nitrite. -Galactosidase levels in cells adapted to the indicated oxyanion levels were monitored.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Effect of increasing amounts of nitrite on narG-lacZ expression in the presence of 1 mM nitrate. The medium additions were as described for Fig. 5.

Comparison of napF-lacZ and narG-lacZ gene expression in batch culture and continuous culture. Prior studies of napF and narGHJI operon expression in response to nitrate and nitrite additions had been done only with batch cell culture. However, it is evident from the chemostat studies described above that considerable regulatory information was not revealed by the batch culture studies. To directly compare the two methods, the same napF-lacZ and narG-lacZ reporter strains used in the continuous culture studies were also analyzed in batch culture (Table 1). For the narG-lacZ strain grown in the presence (40 mM) or absence of nitrate, the patterns of gene expression were similar, although the absolute levels of -galactosidase were higher (by ca. twofold) during chemostat culture. This minor difference may be due to cell growth rate differences between the two methods. We previously demonstrated that narG-lacZ expression is mildly affected by cell growth rate (25).

What nitrate level can E. coli sense? It is apparent that submicromolar to micromolar levels of nitrate are sufficient to induce both narG-lacZ and napF-lacZ gene expression. When the levels of nitrate remaining in the vessel (Fig. 2) were compared to the gene expression levels (Fig. 1A), induction of both reporter fusions was found to occur at a nitrate level below that detected by the analytical methods used here (below 0.03 µM nitrate). The E. coli nitrate two-component regulatory system, composed of the narX, narQ, narL, and narP gene products, must therefore be extremely sensitive to nitrate as an environmental signal. Nitrite, the product of nitrate reduction, appears to play a minor role in regulating the two nitrate reductase operons, since the effect elicited by millimolar levels of this anion were inferior to that elicited by micromolar levels of nitrate. Future studies to examine the roles of the two sensor-transmitter proteins, NarX and NarQ, and the two response regulators, NarL and NarP, should be informative concerning differences in the abilities of the two sensor-transmitter proteins and the two response regulator proteins to modulate nitrate-dependent induction of the narGHJI and the napF operons.

The narGHJI and napF operons are expressed in a complementary style. From the chemostat gene expression studies, we must conclude that the two nitrate reductases in E. coli, encoded by the napF and narGHJI operons, must serve quite different purposes in the cell. When the nitrate level is very low, the cell induces the "low-substrate" nitrate reductase encoded by napF operon to consume nitrate from the environment. The NapF nitrate reductase may therefore be predicted to have a higher (i.e., stronger) affinity for nitrate than does the narGHJI-encoded nitrate reductase. The V_max value might also be predicted to be lower than that of the NarG enzymes. Tests of these predictions must await the development of suitable assays for the biochemical purification and characterization of the NapAB enzyme.

Regulatory implications. From the chemostat studies it is apparent that the nitrate signal transduction system in E. coli is operative when the nitrate concentration in the culture medium (i.e., outside the cell) is in the submicromolar range (Fig. 2). This implies that one or both of the sensor proteins, NarX or NarQ, can detect this low concentration of nitrate in the environment. Greater than 100- to 1,000-fold higher levels of nitrite are needed to give lower levels of narG and napF induction (this study). Therefore, the prior model of Stewart and coworkers (10, 23) for nitrite- and nitrate-dependent gene control by the Nar regulon needs to be revised to account for the ability of the cell to discriminate between these two anions. The prior model for napF control states that nitrite is superior to nitrate as an inducer signal, because any nitrate present in the cell environment would lead to inactivation of NarL-phosphate via NarX cophosphatase activity. In contrast, nitrite presumably does not elicit this effect, so that nitrite is a better inducer of napF gene expression. However, the chemostat data invalidate these viewpoints. Studies to resolve the individual contributions of the NarL and NarP proteins at low substrate levels to the activation and repression of napF gene expression are in progress.