INTRODUCTION

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Many bacterial genes and some genes in eucaryotes are regulated by pairs of regulatory proteins that belong to the two-component class of signal-transducing proteins. The larger of the pair, a sensor kinase, responds to an environmental signal to control expression. In the simplest type of this system, the sensor kinase is autophosphorylated on a histidine residue, which is followed by phosphotransfer to an aspartate residue of the smaller protein, the response regulator, whose phosphorylated form stimulates transcription (for a review, see reference 14). In addition to the autokinase and phosphotransferase of the sensor kinase, two-component systems also have a phosphatase activity to dephosphorylate the response regulator and thereby terminate the response. In some cases, the phosphatase activity is mediated by the sensor kinase, while in others it is mediated by a separate protein or is an inherent property of the response regulator. Among the major questions about two-component regulators is the nature of the signal to which they respond. The signal is well established for some but is either unknown or controversial for others.

In this study, we addressed the nature of the signal that the KdpD sensor kinase and the KdpE response regulator sense. These two proteins control expression of the Kdp transport system of Escherichia coli, a P-type transport ATPase with high affinity for K⁺ (for a review, see reference 1). The Kdp transport system and its two regulatory proteins are found in many bacteria, both gram negative and gram positive. Kdp is one of three saturable K⁺ uptake systems in E. coli, the one with the highest affinity for K⁺ (4). Under most conditions, the other two K⁺ transport systems, Trk and Kup, which are expressed constitutively, satisfy the cells' need for K⁺ and Kdp is not expressed. However, when the concentration of K⁺ ([K⁺]) in the medium drops sufficiently, Kdp is expressed.

Experiments performed with a transcriptional kdp::lacZ fusion led to the hypothesis that expression of Kdp occurred when turgor pressure was suboptimal (15). The fact that Kdp is expressed when the medium [K⁺] is low suggested that the concentration itself was sensed. However, this idea was contradicted by the finding that the [K⁺] below which Kdp was expressed depended on the K⁺ transport systems present. In a strain with both the Trk and Kup systems, expression of Kdp occurred only in medium containing less than 2 mM K⁺; in a mutant lacking all saturable systems, expression was seen in media containing a 50 mM or lower [K⁺]. Other experiments showed that the internal [K⁺] does not determine expression either, since when the internal [K⁺] was varied by changing medium osmolarity, expression could be turned on or off by varying the external [K⁺]. The fact that reducing the medium [K⁺] could turn on expression of Kdp while the specific [K⁺] below which expression occurred depended on the medium and the strain suggested it was not K⁺ per se but the cell's need for K⁺ that was sensed.

The quantitative requirement for K⁺ is its role as a cytoplasmic osmotic solute to maintain turgor pressure. Hence, it was proposed that the need for K⁺ was sensed by changes in turgor pressure, with reduced turgor pressure resulting in expression of Kdp. This model was supported by showing that an osmotic upshock, which transiently reduced turgor pressure under conditions in which neither the internal nor the external [K⁺] was reduced, resulted in a transient burst of high-level expression of Kdp (15).

Subsequent studies have raised questions about the turgor model for control. Altered expression of Kdp was observed in medium with a constant [K⁺] under several conditions. When the medium pH was reduced, Kdp expression increased (2). When the osmolarity of the medium was increased by salt, Kdp expression was higher than when a similar increase in osmolarity was produced by a nonionic solute (2, 13, 22). It was also found that addition of the compatible solute glycine betaine (betaine), expected to reduce Kdp expression because it replaces part of the cell K⁺ pools, did not reduce expression (2).

A major difficulty in testing the turgor model is the absence of reliable methods to measure turgor pressure in E. coli and most bacteria. The only direct method, the collapse pressure of gas vesicles, is restricted to species that produce these flotation structures (24). The only firm statements about changes in turgor pressure that can be made deal with transient changes: an abrupt increase in medium osmolarity will reduce turgor, and a decrease in medium osmolarity will increase turgor. These changes last only until the cells adapt to restore turgor. During steady-state growth, no firm statements about turgor can be made. Hence, all data about Kdp expression during steady-state growth are based on indirect inferences or assumptions about effects of manipulations on turgor.

We re-examined Kdp regulation in the steady state under conditions that were believed to argue against control by turgor. In these studies, we also measured the growth rate, a parameter not included in some of the work that raised questions about the turgor model. We refer to the threshold [K⁺] as that where the growth rate starts to fall as [K⁺] is reduced. The results show a good correlation between the threshold [K⁺] and the onset of expression of Kdp. This correlation had been observed earlier in experiments in which growth rates were measured (15). A simple and parsimonious interpretation explains the drop in growth rate under these conditions as due to reduced turgor. Although other factors seem to modulate, to some extent, the quantitative level of expression, our steady-state growth data are consistent with the hypothesis that a reduction of turgor pressure is necessary and sufficient for expression of Kdp.

	MATERIALS AND METHODS

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Strain TK2469 (F rha thi nagA (lac)179 trkD1 (trkA) (kdpA24'-lacZYA) and its derivatives were used in this work. TK2469 is defective in all three saturable K⁺ transport systems (Kdp, Trk, and Kup) and carries a stabilized transcriptional kdp::lac fusion (18). A Kup⁺ (TrkD⁺) derivative, TK2486, was used in the experiment of Fig. 2, and a Trk⁺ (TrkA⁺) derivative, TK2470, was used for the experiments of Fig. 5. Other derivatives of TK2469 used in particular experiments are described later in the text.

Cells were grown at 37 ± 2°C in the phosphate-buffered minimal medium described earlier (15). In all experiments, [K⁺] was varied without changing osmolarity by replacing K⁺ with equimolar concentrations of Na⁺ or arginine. Media of different pHs were prepared by varying the relative concentrations of mono- and dibasic phosphates while keeping osmolarity constant. The total salt concentrations (K⁺ plus Na⁺) were 119 mM in pH 7.5 medium and 105 mM in pH 6.3 medium in the experiments of Fig. 1 and 2.

Osmotic solutes were added to standard medium containing 70 mM PO₄. The osmotic solute used was either 0.5 M glucose, 0.3 M salt representing a mixture of NaCl and KCl, 0.3 M salt representing a mixture of arginine-HCl and KCl, or 0.2 M salt representing a mixture Na₂SO₄ and K₂SO₄. Expression of Kdp in media of high osmolarity using sulfates or arginine was measured only in the absence of betaine.

Cells were grown to mid-log phase in medium with the highest [K⁺] and then diluted directly (or after washing by centrifugation and suspension in medium containing no K⁺) to tubes of the desired [K⁺] at a density of 4.5 × 10⁷ to 8 × 10⁷/ml. Measurements of growth rate and enzyme level are averages over the subsequent 1.5 to 3 doublings, with the least growth observed in the tubes with the lowest [K⁺].

Growth rates were determined from periodic measurements of culture turbidity at 610 nm in a Bausch & Lomb Spectronic 20 colorimeter. Activity of -galactosidase was assayed at 28°C as previously described after permeabilization of the cells with toluene (11). Assays were done routinely in duplicate but in triplicate for the data of Table 2 and in quadruplicate for the data of Fig. 5; average values are shown. The K⁺ transport measurements of Table 1 were performed at 30°C on cells grown at 30°C as described earlier, either in cells depleted of K⁺ by treatment with 2,4-dinitrophenol (19) or by stimulating uptake in growing cells by an osmotic upshock (20). The net uptake data of Table 2 are the rate of increase of cell K⁺ content during growth, the product of the average content of K⁺, and the growth rate constant. Duplicate measurements of K⁺ transport almost always agreed within 15%, so we estimate that the standard deviation of such measurements is 10% of the measured value or less. The dry weight of cells was estimated by cell turbidity and a calibration curve relating turbidity to the dry weight of cells washed twice by centrifugation with 50% ethanol and then dried to constant weight.

	RESULTS

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Effects of pH on expression of Kdp. The dependence of growth rate and Kdp expression on medium [K⁺] is shown in Fig. 1 for media of two different pH values: open symbols are data for pH 6.3 medium, and filled symbols are data for pH 7.5 medium. At each pH, growth was rapid and Kdp expression was at a low basal level at the highest [K⁺]. As the [K⁺] was reduced, there came a point where Kdp expression began to rise and the growth rate fell. To be able to show the wide range of Kdp expression, up to almost 1,000-fold in these experiments, the expression data are shown on a logarithmic scale. The data of Fig. 1 show that Kdp expression began at a higher medium [K⁺] in low-pH medium than in high-pH medium. These results are in agreement with those of Asha and Gowrishankar (2), who reported that the magnitude of Kdp expression was inversely correlated with the [K⁺] required for half-maximal growth. Our data extend this correlation to show that expression above the basal level begins at the threshold [K⁺] at which the growth rate began to decline. Hence, expression correlates with the point at which the cells' needs for K⁺ are no longer fully satisfied and implies that it is the K⁺ need, not pH per se, that accounts for Kdp expression. A higher [K⁺] requirement for growth at low pH was noted for strain TK405 [pertinent genotype, (kdpFAB)5 trkD1 trkA405], whose K⁺ transport properties are like those of strain TK2469, in an early report on K⁺ transport mutants (19).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Effect of medium pH and [K+] on kdp expression in strain TK2469. Cells were grown, and average growth rates and -galactosidase activities were measured as described in Materials and Methods. The scale of -galactosidase activity is logarithmic to show the full range of values obtained. Open symbols are for growth at pH 6.3; closed symbols are for pH 7.5. The control (100%) growth rates were 0.65 h1 at pH 6.3 and 0.81 h1 at pH 7.5.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Effect of medium pH and [K+] on kdp expression in Kup+ strain TK2486. Data were obtained and plotted as described for Fig. 1; open symbols are for pH 6.3, and closed symbols are for pH 7.5. The control (100%) growth rates were 0.87 h1 at pH 6.3 and 0.88 h1 at pH 7.5.

Roles of compatible solutes in expression of Kdp. The effects of the compatible solute betaine on Kdp expression and growth are shown in Fig. 3 and 4 for strain TK2469, which requires a high [K⁺] for growth in all media. Since betaine is accumulated to high levels only in high-osmolarity medium, these experiments were done with medium containing either 0.3 M salt or 0.5 M sugar, creating virtually equivalent medium osmolarity. In the cultures without betaine (open symbols), Kdp expression began to rise above the basal level while the growth rate remained unchanged but did not rise above twice the basal level until the growth rate began to fall. In the presence of betaine, the results were very similar, except that the growth rate under all conditions was higher. The increase in growth rates at the highest [K⁺] were 28% in medium with salt and 50% in medium with sugar. The relative growth rate increase produced by betaine was larger as the growth rate became limited by a lower [K⁺]. Expression of Kdp was somewhat higher in the presence of betaine, but the increase was less than twofold in medium with salt and less than threefold in medium with sugar. These results are in good agreement with those of Asha and Gowrishankar (2), who noted a modest increase of expression in the presence of betaine.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Effect of betaine and [K+] on kdp expression and growth rate in strain TK2469 grown in medium containing 0.3 M added salt. Data were obtained as described in Materials and Methods and plotted as described for Fig. 1. Open symbols are for the absence of betaine, and filled symbols are for the presence of 2.5 mM betaine. The control (100%) growth rate was 0.57 h1.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Effect of betaine and [K+] on the kdp expression and growth rate of strain TK2469 grown in medium containing 0.5 M added glucose. Data were obtained as described in Materials and Methods and plotted as described for Fig. 1. Open symbols are for the absence of betaine, and filled symbols are for the presence of 2.5 mM betaine. The control (100%) growth rate was 0.45 h1.

Greater effectiveness of salt in expression of Kdp. Comparison of Fig. 3 and 4 shows that Kdp expression begins at a much higher [K⁺] in medium in which NaCl was the osmotic solute than when glucose was used. A similar difference was seen in strain TK2470, a Trk⁺ derivative of TK2469 which has a much lower K⁺ requirement for growth (Fig. 5). Data for growth with the two types of osmotic solutes are shown in the same figure to highlight the greater effectiveness of salt. These results confirm considerable data in the literature showing that a salt such as NaCl provokes greater expression of Kdp than do osmotically equivalent concentrations of a sugar such as glucose. However, the differential effect of salts is limited to intermediate [K⁺]s: at a sufficiently high [K⁺], Kdp is not expressed in either medium, while at a sufficiently low [K⁺], Kdp is expressed to high levels in both media. A high level of expression in the NaCl medium occurs only at a [K⁺] at which substantial expression occurs in the glucose medium.

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Effects of salt and sugar as osmotic solutes on the dependence of growth rate and Kdp expression on [K+] in Trk+ strain TK2470. Cells were grown in medium containing 0.3 M added salt (filled symbols) or 0.5 M added glucose (open symbols). Data were obtained as described in Materials and Methods and plotted as described for Fig. 1, except that the data for -galactosidase are averages of four determinations and growth data are averages of two determinations. The control growth rate in the salt medium () was 0.70 h1, while that in the glucose medium () was 0.65 h1.

Alterations in K⁺ transport correlate with effects on growth and Kdp expression. The K⁺ requirements for growth of mutants defective in K⁺ transport were found to correlate with the degree to which K⁺ transport was impaired (19). We found the same correlation here for strain TK2469 for the effects of pH shown in Fig. 1 and the greater effectiveness of salts than sugar in Fig. 3 and 4. The greater K⁺ requirement of strain TK2469 at low pH is explained by the marked pH dependence of K⁺ uptake in this strain (Table 1). Transport at pH 6.7 was only 26% of the rate at pH 7.43. The greater inhibition of growth and expression in strain TK2469 by high concentrations of salt is reflected in a greater inhibition of K⁺ uptake by salt (Table 1). The rate of uptake provoked by osmotic upshock with NaCl was only 39% of that when glucose was used as the osmotic solute and rose only slightly when upshock was performed with equal mixtures of salt and sugar.

1

1

Roles of growth rate and net K⁺ uptake. We examined the correlation of growth rate and of net K⁺ uptake on expression of Kdp by comparing cells growing on DL-lactate, where the growth rate is slower, with those growing on glucose. In lactate medium, the overall pattern of expression is like that in Fig. 1, with the exception that both the basal level and the highest level of expression were lower than during growth on glucose. Table 2 shows data for growth rate, Kdp expression, and net K⁺ uptake for a few [K⁺]s during growth on lactate or glucose. These measurements were made to determine if there is a direct effect of either growth rate or net K⁺ uptake on expression of Kdp. Cells growing on lactate in 70 mM K⁺ medium had a relatively low rate of growth and of net K⁺ uptake yet did not express Kdp above the basal level. In contrast, cells growing on glucose in medium with either 40 or 44 mM K⁺ had a higher rate of growth and of net K⁺ uptake yet expressed Kdp at 100 to 200 times the basal levels. The data show that neither the rate of growth per se nor the rate of net K⁺ uptake correlates with expression of Kdp; limitation of growth rate by the medium [K⁺] does correlate.

Inhibition of growth in Kdp⁺ Trk strains. In a strain like TK2469 but with a wild-type copy of Kdp, it was found that expression of Kdp, as monitored by the fusion, was highest at 1 mM K⁺ with reduction at high concentrations so that expression resembled that in TK2469, except that the expression level was much lower (15). A recent report of Kdp expression in an analogous strain of Salmonella typhimurium confirmed the expression but noted that growth of the strain was inhibited up to 30% as the K⁺ was increased from 1 to 25 mM (12). The reduction in growth rate was puzzling but viewed as an apparent contradiction to control by turgor. We found the same growth phenomenon in a corresponding strain of E. coli (TK2469 with F-100 carrying wild-type kdp genes), as well as in a Kdp⁺ transductant of TK2469 (where the fusion is replaced by wild-type kdp genes), indicating that this effect is neither unique to Salmonella nor an artifact of the kdp::lac fusions. Growth rates are high at low (1 mM) and high (100 mM) [K⁺]s but reduced over the range of about 5 to 50 mM with maximum reduction at 20 to 30 mM K⁺. However, when such strains are grown in minimal medium in which aspartate replaces ammonium as the nitrogen source, growth rates are essentially independent of medium [K⁺] over the range of 1 to 100 mM. These results suggest that growth inhibition is an inhibitory effect of ammonium ions, which are inhibitory only when Kdp is expressed and the medium K content is not low. Inhibition could be due to ammonium uptake by Kdp, since it has been reported that ammonium ions are substrates of the Kdp system (6).

	DISCUSSION

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Kdp expression is correlated with K⁺ limitation of growth. We examined expression of Kdp and growth rate as a function of [K⁺] under conditions that were believed by some to be inconsistent with the control of Kdp expression by turgor. The marked effect of pH on the expression of Kdp (Fig. 1) is a reflection of the increased K⁺ requirement for growth at low pH. The fact that there is an inverse correlation between growth rate and Kdp expression was noted by Asha and Gowrishankar (2), but that statement could easily be overlooked since it was in the context of a paper that marshalled arguments against control by turgor. We confirm here the correlation noted before and, further, show that Kdp expression begins to rise near the threshold [K⁺] for growth. In a strain whose growth is less sensitive to pH, expression of Kdp is also less pH dependent (Fig. 2).

Effects of compatible solutes. We have confirmed the seemingly anomalous effects of betaine on Kdp expression and shown that they fit the correlation with K⁺ limitation. The result is readily reconciled with what is established about the roles of compatible solutes in enteric organisms. Such solutes are synthesized or accumulated only during growth in media of elevated osmolarity and serve to replace part of the pools of K⁺ and glutamate that are necessary for osmotic equilibrium in the absence of compatible solutes. The increase in rate of growth produced by compatible solutes is presumably due, at least in part, to the replacement of the high concentrations of ionic solutes that are inhibitory to many enzymes with neutral molecules that inhibit enzymes little, if at all, even when present at high concentrations.

Other factors in the expression of Kdp. Our data suggest that factors in addition to turgor modulate the expression of Kdp. Addition of betaine increases Kdp expression nonspecifically up to threefold, both when expressed at a basal level and when induced. This stimulation indicates that the cytoplasmic environment, specifically, the solutes present, has a general influence on the relative expression of genes. There must be other genes whose expression is higher in the absence of betaine.

The flux model. A number of other models for control of Kdp have been proposed, but the only one that seems consistent with the data and testable is the K⁺ flux model of Asha and Gowrishankar (2). Their proposal is that Kdp is expressed when K⁺ influx falls below some particular level. The flux model has one great advantage over the turgor model: it assumes sensing by a parameter than can be measured, which is not the case for turgor.

KdpD is a plausible sensor of turgor. The known sensor for Kdp expression, the KdpD protein, has a structure and features that make it a plausible candidate as a turgor sensor. KdpD has four membrane spans that are important in its sensing function (23, 25). A deletion variant lacking the membrane spans retains the ability to express Kdp at a low level but has lost the ability to respond to the environment (17). Mutations in and near the membrane spans result in partial constitutive expression of Kdp (21). Turgor, sensed as a stretch in the plane of the inner membrane, could alter the structure of the membrane spans, perhaps causing rotational movement that is transmitted to the kinase domain in the C terminus of KdpD.