INTRODUCTION

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During anaerobic growth, Escherichia coli is capable of synthesizing two uptake hydrogenase isoenzymes, Hyd1 and Hyd2, which catalyze the oxidation of hydrogen gas (5, 32). Hyd1 and Hyd2 are heterodimers, consisting of a small subunit and a nickel-containing catalytic large subunit (5, 32), that localize to the inner membrane facing the periplasmic space (28, 29). The H₂ uptake-specific activity of purified Hyd2 is greater than that of Hyd1, while purified active Hyd1 is more resistant to denaturation by changes in pH and prolonged exposure to oxygen (5, 32). The pH optimum for Hyd1 ranges from 6 to 8, whereas that of Hyd2 is 8 (5, 32).

The genes encoding the Hyd1 small and large subunits, hyaA and hyaB, respectively, comprise part of the hya operon (24), while the genes encoding the Hyd2 small and large subunits, hybA and hybC, respectively, comprise part of the hyb operon (23). In addition to the structural genes, the hyaDEF and hybFG genes are required for posttranslational modification of nascent structural subunits (23, 25). Complete transcription of both operons is required for maximal expression of active Hyd1 and Hyd2 complexes (23-25).

The redundancy of uptake hydrogenase formation is partially explained by the differential regulation of hya and hyb operon expression and functions of Hyd1 and Hyd2 in anaerobic metabolism. Anaerobic induction of hyb expression requires the Fnr protein and is enhanced by exogenous fumarate (31, 40). Likewise, Hyd2 activity levels are maximal in cultures grown on hydrogen and fumarate (23, 31). An hyb mutant that lacks Hyd2 activity is unable to grow anaerobically on hydrogen and fumarate (23), which strongly suggests that Hyd2 functions in contributing electrons from hydrogen oxidation to fumarate reductase for the reduction of fumarate to succinate (5, 23, 31).

Hyd1 has no defined function in anaerobic metabolism, but it has been proposed to shuttle electrons from formate to fumarate during fumarate reduction (12) and/or to oxidize hydrogen gas and contribute electrons to the quinone pool (31). Transcription of hya is induced by anaerobiosis and repressed by nitrate (9) and requires the sigma factor RpoS (2) and the transcriptional regulators ArcA and AppY (9). Conflicting reports on the effects of carbon source on hya expression and Hyd1 synthesis during growth have been published. Sawers et al. reported that Hyd1 enzymatic activity was maximal in cultures grown on glucose and enhanced by the addition of exogenous formate (31). Formate also led to the recovery of Hyd1 activity in a pfl-1 mutant defective in formate production (17, 31). Furthermore, Bronsted and Atlung demonstrated that formate enhanced the level of hya transcription during fermentative growth on glucose (9). In contrast, Menon et al. have reported that the levels of active Hyd1 are essentially identical in cultures grown on either glucose or glucose plus formate (25).

Although the physiological function of Hyd1 has not yet been defined, the response of hya expression to carbon and phosphate starvation (2) and dependence on RpoS and AppY (2, 9) suggest that it is involved in response to stress. Here we report that during anaerobic growth, hya responds to the external pH (pH_e). We also determined the effect of pH_e on the expression of hyb to characterize the effect of pH_e on anaerobic expression of both uptake hydrogenase operons.

	MATERIALS AND METHODS

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Bacterial strains. The bacterial strains used in these experiments are listed in Table 1. All generalized transductions were conducted as described previously (34) with phage P1_vir. To create strain JK22, the selectable marker zjj::Tn10 from donor strain ECL618 (14) was transduced into strain JK2. The resulting Ap^r Tet^r transductants were then scored for sensitivity to toluidine blue, as described previously (15), to indicate inheritance of the arcA2 mutation.

Construction of the hya'-'lacZ chromosome fusion and strain JK2. Plasmid pCL47 (24) containing the hya promoter and the entire hya operon was digested with EcoRI and BamHI. The 470-bp EcoRI-BamHI fragment containing the promoter region and first 164 bp of hyaA was isolated by agarose gel electrophoresis, purified, treated with EcoRI and BamHI, and ligated into EcoRI-BamHI-digested plasmid pMC1403 (Ap^r, 'lacZYA) (11). This created an in-frame fusion of codon 58 of hyaA with codon 8 of lacZ. Plasmid pAF11 was isolated by its ability to confer Lac⁺ Ap^r to BW545, and the presence of the hya'-'lacZ junction was confirmed by DNA sequencing.

Growth conditions. Strains were routinely cultured at 37°C in liquid Luria broth (LB) (10% tryptone, 5% yeast extract, 10% NaCl) or on solid Luria agar medium (LB with 15% agar added). Carbon sources and supplements for growth in liquid media were used at the following concentrations: glucose, 0.4%; glycerol, 0.4%; sodium fumarate, 0.5%; sodium formate, 0.4%. Growth in liquid minimal media was performed with M63 medium (34) with 0.2% glucose as a carbon source. Antibiotics were used at the following concentrations: 100 µg of ampicillin per ml, 30 µg of kanamycin per ml, and 25 µg of tetracycline per ml. Cultures were grown aerobically by shaking 20-ml cultures in 250-ml flasks at 250 rpm on a rotary shaker. Anaerobic growth of cultures was carried out in either 15-, 20-, or 35-ml bottles fitted with a rubber stopper and capped (25).

Anaerobic expression of hya'-'lacZ and hyb'-'lacZ fusions. To assay for either hya'-'lacZ or hyb'-'lacZ expression in cells, the appropriate strain was grown aerobically overnight in liquid medium and diluted 1:100 in 20 ml of LB or M63 medium (pH 7.2) containing either glucose (hya'-'lacZ expression assays) (25) or glycerol and fumarate (hyb'-'lacZ expression assays) (23). This culture was grown aerobically until mid-log phase (approximately 2 to 3 h). A 1-ml volume of cells was collected by centrifugation, washed, and resuspended 1:1 in fresh medium. These cells were then diluted 1:100 in 20 ml of either LB or M63 medium containing the appropriate carbon source and grown anaerobically as described above.

pH_e shift experiments. To measure the response of hya'-'lacZ expression to a shift from alkaline to acidic pH_e, an aerobic culture of strain JK2 was prepared as above and used to inoculate 35 ml of LB containing glucose and buffered at pH 7.8 with 10 mM TES. This culture was grown anaerobically. Samples to determine pH, optical density at 600 nm (OD₆₀₀), and -galactosidase specific activity were taken at 15-min intervals for approximately two generations of growth (OD₆₀₀ = 0.2). At this point, 15 ml of the culture was removed with a syringe and added to a prewarmed 15-ml stoppered bottle containing 1.0 M MES (pH 4.0) to give a final concentration of 63 mM and a pH of 5.8. Samples from both cultures were then taken at the time of the shift and then every 15 min for at least 2 h.

-Galactosidase activity assay, pH_e determination, and measurement of optical density. The cells from 1-ml culture samples were collected immediately by centrifugation, and the pH of the supernatant was determined. The cell pellet was resuspended in a 1-ml volume of Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄ · 7H₂O, 50 mM -mercaptoethanol; buffered at pH 7.0) (26) and kept at 4°C until assayed. Cell suspensions were assayed spectrophotometrically for the OD₆₀₀. -Galactosidase specific activity was assayed in duplicate as described previously (26) and measured spectrophotometrically by using the following calculation: 1 Miller unit = 1,000 × (OD₄₂₀  1.75 OD₅₅₀)/time (minutes) × 0.1 ml × OD₆₀₀ (26). Specific activities are the average of the values obtained in the two assays. Each experiment was performed at least twice. All OD measurements were conducted with a Cary 2000 spectrophotometer; the culture pH was measured with a Radiometer PH62M pH meter.

	RESULTS

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Response of hya expression to pH_e. We initially cultured strain JK2 (attB::hya'-'lacZ) anaerobically in unbuffered LB containing glucose to monitor hya expression during growth (Fig. 1). The onset of hya expression was concurrent with the decline of the medium pH. Cultures began to express hya as the pH_e began to decline. As cultures progressed in growth and the pH_e continued to decline, hya expression continued to increase and eventually peaked in late stationary phase when the pH_e had fallen to 4.7. 

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Time course of hya expression during anaerobic growth. Strain JK2 was cultured in unbuffered LB with glucose and assayed for -galactosidase specific activity (Miller units) (solid circles) and OD600 (open circles). pHe values of selected samples are indicated.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Effect of RpoS and ArcA on the response of hya expression to external pH. Cultures were grown in either LB (solid lines) or M63 medium (dashed lines) containing glucose and buffered at either pH 5.5 (circles) or pH 8.5 (triangles). Time point samples were assayed for -galactosidase specific activity (Miller units) (solid symbols) and OD600 (open symbols). (A) Strain JK2 (hya'-'lacZ). (B) Strain JK1222 (rpoS::Tn10 hya'-'lacZ). (C) Strain JK22 (arcA2 hya'-'lacZ).

Effect of a pH_e shift on hya expression. To test if the hya response to pH_e could be initiated by a more rapid change in the pH_e value, anaerobic cultures were grown to the point of early exponential phase and then the pH_e value was shifted. As demonstrated in Fig. 3A, a shift to an acidic pH_e value stimulated hya expression. The level of hya expressed in acidic versus untreated cultures was 6-, 13-, 70-, and 20-fold higher at 15, 30, 45, and 60 min postshift, respectively. A slight effect on the growth rate was observed in the treated culture (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Effect of a shift in pHe on hya expression. Growth of JK2 cultures was initiated in buffered LB containing glucose. At the point in growth indicated by the arrow, the cultures were split and buffer was added to one portion to achieve a shift in pH value. The cultures were monitored for -galactosidase specific activity (Miller units) (solid symbols) and OD600 (open symbols). (A) Cultures grown in medium at pH 7.8 (10 mM TES) (triangles) and shifted to pH 5.8 (circles) by the addition of 1 M MES (pH 4). (B) Cultures grown in medium at pH 5.8 (10 mM MES) (circles) and shifted to pH 7.8 (triangles) by the addition of 1 M TES (pH 8).

Effect of Pfl activity and formate on hya expression. In E. coli, the enzyme pyruvate formate-lyase (Pfl), encoded by the pfl gene, is responsible for the production of formate from pyruvate during fermentative growth (19). Initially, excess formate is excreted into the culture medium (7), and then, as the pH_e declines, formate is transported back into the cell (30) and further metabolized (27).

Response of hya expression to pH_e in the absence of RpoS. In E. coli, the levels of the sigma factor RpoS increase in response to conditions of stationary-phase growth (13), acidic pH_e (6), and increasing concentrations of fermentation end products (33). We and others (2) have noted that RpoS is required for maximal expression of hya; thus, it was possible that the change in hya expression in response to pH_e results from a change in RpoS-mediated expression. Therefore, we tested the response of hya to pH_e in the absence of RpoS by culturing an RpoS strain in either acidic or alkaline medium (Fig. 2B). In the absence of RpoS, the response of hya expression to pH_e was not lost. The expression levels, however, were reduced compared to expression in wild-type cultures (compare Fig. 2B with Fig. 2A). In RpoS cultures, maximal hya expression levels were achieved in early stationary phase and then rapidly declined, whereas in wild-type cultures, maximal expression was sustained in the stationary phase for a longer period (compare Fig. 2B with Fig. 2A). Growth of RpoS cultures in acidic medium led to a fourfold increase in maximal expression levels over levels achieved in alkaline medium (Fig. 2B). There was no significant difference in pH_e values during growth of RpoS cultures compared to wild-type cultures (data not shown).

Response of hya expression to pH_e in the absence of ArcA. Like RpoS, ArcA is required for full expression of hya (9). A putative signal for ArcA activation is a change in proton motive force (p) (8, 16), and one component of p is pH_e. Thus, the response of hya expression to pH_e could involve ArcA. This possibility was tested by examining the hya response to pH_e in the absence of ArcA (Fig. 2C). In both acidic and alkaline media, hya expression levels were reduced in ArcA cultures compared to wild-type cultures (compare Fig. 2C with Fig. 2A). The difference in the onset of expression observed in wild-type cultures grown at the two pH_e values was diminished in ArcA cultures. Thereafter, as cultures progressed into stationary phase, the absence of ArcA abrogated elevated hya levels in acidic medium. Interestingly, ArcA cultures expressed 1.6-fold higher levels in alkaline medium than in acidic medium. Again, no significant difference was observed in the values of pH_e for ArcA and wild-type cultures (data not shown).

Expression of hya in response to pH_e in the absence of AppY. The transcriptional activator AppY is required for full expression of hya during anaerobic growth (2, 9). The response of hya expression to pH_e was tested in the absence of AppY to determine whether AppY was involved in the response of hya to pH_e (Fig. 4). The onset of hya expression in acidic medium occurred at the same point during growth of cultures without AppY as in wild-type cultures. However, in cultures without AppY, hya expression plateaued after only 1 h of growth whereas in wild-type cultures, hya expression continued to increase for an additional 6 h. In alkaline medium, the onset of hya expression in both strains was inhibited until entry into stationary phase. As above, wild-type cultures expressed higher levels in acidic than in alkaline medium whereas in AppY cultures, maximal levels were 3.5-fold higher in alkaline than in acidic medium. In alkaline medium, the pH_e value of AppY cultures dropped to a neutral value 4 h later during growth than did the pH_e value of wild-type cultures (data not shown); this was the same point in growth where hya expression in AppY cultures reached maximal levels.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Effect of AppY on the response of hya expression to external pH. Cultures were grown in LB containing glucose buffered at pH 5.5 (circles) or 8.5 (triangles). Time point samples were assayed for -galactosidase specific activity (Miller units) (solid symbols) and OD600 (open symbols). (A) Strain JK2193 (hya'-'lacZ). (B) Strain JK256 (appY hya'-'lacZ).

Expression of hyb in response to pH_e. Anaerobic growth of E. coli cultures results in the synthesis of both Hyd1 and Hyd2 uptake hydrogenases. To further characterize anaerobic uptake hydrogenase operon expression in response to pH_e, we studied the expression of a hyb'-'lacZ fusion in both acidic and alkaline media containing glycerol and fumarate (Fig. 5). The onset of hyb expression in cultures was constitutive with respect to the pH_e value and occurred 30 min after the initiation of anaerobic growth. In contrast to hya expression, hyb expression levels in acidic medium achieved maximal values after only 2 h of growth whereas expression in alkaline medium continued to increase during the entire 8 h of growth. Consequently, maximal hyb expression levels attained in alkaline medium were fourfold higher than in acidic medium.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Response of hyb expression to external pH during growth. Strain SE2065 containing the hyb'-'lacZ chromosome fusion was cultured in LB containing glycerol and fumarate buffered at pH 5.5 (circles) or 8.5 (triangles). Time point samples were assayed for -galactosidase specific activity (Miller units) (solid symbols) and OD600 (open symbols).

	DISCUSSION

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The difficulty in resolving the function of Hyd1 is partially due to the complexity of the way in which hya expression is regulated (2, 9). Anaerobic growth induces hya expression; however, E. coli does not require Hyd1 for anaerobic growth. Limitation of carbon and phosphate contributes to anaerobic expression (2), but the effect on Hyd1 synthesis is unknown. Another possible source of stress is acidification of the external environment (6), and as we have shown in this study, anaerobic expression of hya responds to pH_e and is maximal at an acidic pH_e. This response of hya was evident in both complex and minimal media, suggesting that components found in yeast extract or tryptone, such as amino acids, are not mediating the response. However, the level of expression achieved at alkaline pH_e values was greater in complex medium than in minimal medium. This could reflect an nutritional enhancement and/or the greater acidification of alkaline LB cultures compared to alkaline minimal medium cultures. Regardless, in both minimal medium and LB, pH_e exerts a significant effect on hya expression.

Formate is not required for hya response to pH_e. We found that exogenous formate enhances the level of hya expression, as previously reported (9), but that the degree of enhancement depended on the growth phase. Formate had a stronger influence during the initial stages of expression, but the influence diminished as expression became maximal. It is important to this study, however, that the response of hya expression to pH_e was evident in the pfl mutant; thus, it does not require formate. While formate was not essential for the hya response to pH_e, it was needed to achieve maximal expression levels. Formate is also essential for synthesis of active Hyd1 (31), since the expression of HydA and HypA, which are required to process Hyd1 precursors to active enzymes (17, 31), is formate dependent (21, 22). Thus, formate is essential for maximal hya expression and Hyd1 precursor processing but does not mediate the hya response to pH_e.

RpoS is required to achieve and sustain maximal hya expression but does not mediate the response to pH_e. Although RpoS mediates changes in gene expression in response to pH_e (6) and although maximal hya expression requires RpoS, the response of hya to pH_e did not require RpoS. This is similar to the effect of RpoS on the hya response to phosphate starvation (2). The period of peak expression that occurred in stationary-phase growth in the absence of RpoS was shortened compared to that in wild-type cultures. Therefore, RpoS enhances and sustains hya expression but does not regulate its response to pH_e. The effect of RpoS on hya during stationary-phase growth is in agreement with the increased level of RpoS in stationary-phase cells (13).

hya responds to pH_e in the absence of AppY but at reduced levels. The transcriptional regulator AppY, which is required for full anaerobic expression of hya (9), was not required for the early onset of hya expression in acidic medium but was needed to attain maximal expression levels. AppY had a greater influence on hya in the stationary phase of growth. This could reflect the elevated levels of appY transcription in stationary-phase cultures (10), which could presumably lead to increased AppY stimulation of hya expression.

ArcA is required for hya to respond to pH_e and to achieve maximal expression. Whereas maximal hya expression required AppY, RpoS, and ArcA, the response of hya to pH_e was most strongly affected by ArcA. Without ArcA, hya expression appeared to respond primarily to changes in growth rate and not external pH. ArcA regulates gene expression in response to changes in the respiratory state of the cell (16) and may be activated by the accompanying decline in p (8, 16). Cultures that are growing anaerobically have a lower p value than aerobically growing cultures do (37). When cultures are grown in acidic medium or reach stationary phase, the p decreases further (18, 37). Since the conditions of growth that minimize p also maximize hya expression, it is plausible that the hya response to pH_e is at least partially mediated by ArcA. As a result, anaerobic and pH_e control of hya expression would be linked through ArcA.

hya and hyb respond differentially to pH_e. While both the hya and hyb operons responded to pH_e, the hyb response reached maximal levels in alkaline medium and the hya response reached maximal levels in acidic medium. This expression pattern is consistent with the activity patterns of the two periplasmic uptake hydrogenases. Hyd2 has an alkaline pH optimum (5) and is at its highest levels during early growth phases, when the medium and periplasm are alkaline (23, 36). Hyd1 has an acidic optimum (32) and is at its highest levels during later stages of growth (25), when the pH of the medium and periplasm have declined (36) and the activity of Hyd2 has begun to diminish (23). Thus, the expression of hya and hyb is upregulated by pH conditions which favor the activity of the respective periplasmic enzymes. In support of this, active Hyd1 is present at severalfold higher levels in acidic cultures than in alkaline cultures throughout growth (data not shown). Expression of the hyb operon is also needed for maximal production of active Hyd1 (23), since one of the genes of hyb is required for the efficient processing of Hyd1 (23). Therefore, under acidic conditions when Hyd2 activity becomes less important, expression of hyb is maintained as a component of Hyd1 processing.