INTRODUCTION

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Nitrogen fixation in legume nodules involves a complex exchange of nutrients between the plant and bacteroids. This exchange involves transport across the bacteroid membrane and the plant-derived envelope surrounding the bacteroid, the peribacteroid membrane. In its simplest terms, this symbiosis is often viewed as an exchange of reduced carbon for reduced nitrogen. However, it is clear that optimum nodule function also involves a balanced flow of other nutrients (33). One nutrient that has been shown to be important for this symbiosis is phosphorus. Low phosphorus availability in soils is common and limits legume production worldwide; however, phosphorus metabolism in this plant-microbe interaction has not been well characterized. Given the significant metabolic activity of bacteroids, the phosphorus supply may be critical for optimum symbiotic functioning of bacteroids, and understanding the mechanisms by which bacteroids acquire phosphorus should provide useful information concerning phosphorus exchange between the symbionts and phosphorus flow in the symbiosis.

Phosphate (P_i) uptake has been investigated in various bacteria. In some microorganisms only a single transport system has been found. This is the case for Micrococcus lysodeikticus (19) and for several Rhizobium species (41). In other bacteria, two P_i transport systems have been found. Examples of such bacteria include Escherichia coli (34, 53), Acinetobacter johnsonii (48), and Pseudomonas aeruginosa (26). In each of the latter bacteria, a constitutively expressed low-affinity transport system and a P_i-repressible high-affinity permease have been identified. In E. coli, the low-affinity P_i transport system (LATS) is energized by the proton motive force (p) and consists of a single membrane component (17). In contrast, the high-affinity P_i transport system (HATS) is a multicomponent system consisting of proteins associated with the cytoplasmic membrane, an ATP-binding protein, and a periplasmic solute-binding protein (reviewed in reference 51).

Recently, Sinorhizobium meliloti has been reported to have at least two P_i transport systems, consistent with the high-affinity-low-affinity model described above (49). The high-affinity system is encoded by the phoCDET operon, and the low-affinity system is encoded by pit (in the orfA-pit operon) (6). Previously published evidence strongly suggests that expression of the genes coding for both P_i transport systems in S. meliloti is controlled by PhoB (6). PhoB (presumably phosphorylated PhoB) positively regulates the phoCDET operon but negatively controls orfA-pit. Under nonlimiting P_i conditions, the low-affinity Pit permease is expressed and is primarily responsible for P_i uptake. When S. meliloti is grown under P_i-limiting conditions, the Pit system is repressed, while the high-affinity PhoCDET system is induced and becomes the primary mechanism of P_i transport.

Some of our efforts to characterize and understand phosphorus metabolism and exchange in the Rhizobium-legume association have focused on the Rhizobium tropici-bean symbiosis (1), with initial work aimed at characterizing P_i assimilation and regulation in the microbial partner. As observed with other gram-negative bacteria (51), R. tropici induces alkaline phosphatase, and its P_i transport rate increases significantly in response to P_i limit-limiting conditions (1). The induction occurs when the medium P_i concentration is approximately 1 µM (1). R. tropici bacteroids isolated from nodules of bean plants grown in the presence of nonlimiting phosphorus concentrations contain extremely high levels of alkaline phosphatase, as well as a P_i stress-inducible acid phosphatase (1). This implies that under normal growth conditions a bean plant provides very low levels of P_i to the bacteroids in its nodules. In order to determine the importance of P_i supply for the bean-bacteroid symbiotic system, we are now assessing R. tropici P_i transport systems and estimating their kinetic properties. In this report, the P_i transport systems of R. tropici are described. Like S. meliloti (49), this bacterium has two distinct functional P_i transport systems. However, R. tropici appears to differ from S. meliloti and all other bacteria investigated previously since both P_i transport systems are inducible by P_i stress, are shock sensitive, and are energized by phosphate bond energy. In addition, in this paper we also describe a mutant that lacks high-affinity P_i transport activity.

	MATERIALS AND METHODS

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Strains and culture conditions. Strains CIAT899 and CAP45 were used in all experiments. CIAT899 is the type strain of R. tropici type IIB (29), and CAP45 is a P_i transport mutant derived from CIAT899 (see below). CIAT899 was maintained on the minimal mannitol-ammonium agar (MMNH₄) (pH 7.2) described previously (42, 43). CAP45 was maintained on the same medium, except that -glycerolphosphate (GP) replaced mannitol as the sole carbon source and gentamicin was included at a final concentration of 25 mg · liter¹. The other antibiotics used in the experiments were ampicillin (100 mg · liter¹) and tetracycline (25 mg · liter¹). In experiments in which P_i-starved cells (P_i cells) were used, the cells were incubated in MMNH₄ which lacked added phosphorus but was buffered to pH 7.2 with 5 mM MES (morpholineethanesulfonic acid) and 10 mM MOPS (morpholinepropanesulfonic acid) (MMNH₄-OP) (43).

Mutant isolation. Pho regulatory mutants that are constitutive for the P_i-repressible alkaline phosphatase often also do not express a high-affinity P_i transporter (5; reviewed in reference 51). We used the strategy and methods of Torriani and Rothman (44) to isolate R. tropici mutants that expressed alkaline phosphatase constitutively and then screened these mutants for a P_i transport phenotype. Briefly, R. tropici CIAT899 was mutated with transposon Tn5B22 (40) as previously described (2, 15). E. coli S17-1 (39) was used to mobilize the transposon into CIAT899 in mating mixtures, which were plated onto GP-gentamicin agar. In order to be used as a carbon source, GP must first be dephosphorylated at rates sufficient to supply glycerol for growth. One candidate phosphatase in CIAT899 is alkaline phosphatase (1). However, because of the high concentration of P_i in the medium, growth would require phoA expression under conditions where this gene is normally repressed. Transconjugants were isolated from the GP-gentamicin agar plates by streaking twice to obtain pure cultures, and then constitutive expression of alkaline phosphatase activity was measured by comparing alkaline phosphatase activities in cells grown under high-P_i conditions (MMNH₄ broth) and in cells after incubation under zero-P_i conditions (MMNH₄-OP broth). Periplasm proteins were extracted and alkaline phosphatase was assayed by using previously described methods (1). Subsamples of the mutants were then screened for P_i uptake to identify mutants that were defective in P_i transport.

Transport assays. Early-stationary-phase MMNH₄ cultures were washed twice in MMNH₄-OP and resuspended in MMNH₄-OP to an optical density of 0.60 at an absorbance of 595 nm. To obtain P_i cells, washed cells were incubated in MMNH₄-OP at 30°C for 7 h in order to allow for maximum induction of P_i transport (1). Chloramphenicol (50 mg · liter¹) was then added to stop further protein synthesis. Cells not starved for P_i (+P_i cells) were prepared in the same way except that chloramphenicol was added immediately after washing and the cells were used within 1 h. In preliminary experiments, we found that chloramphenicol did not interfere with P_i transport (results not shown) but did inhibit the synthesis of alkaline phosphatase for at least 5 h. Thus, we concluded that de novo protein synthesis in +P_i cells did not occur during the experiments performed with +P_i cells.

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Phosphate exchange and efflux. The methods of Medveczky and Rosenberg (30) were modified slightly for use with R. tropici. Briefly, P_i cells were loaded for 4 min with [³²P]KH₂PO₄ (either 5 or 400 µM; specific activity, 22.5 µCi · µmol¹) at 30°C and then diluted 100-fold with MMNH₄-OP without unlabeled potassium phosphate (efflux experiments) or with either 25 µM or 2 mM unlabeled potassium phosphate (exchange experiments). At time intervals, 0.5-ml samples were filtered and washed with transport rinse buffer as described above for the transport experiments.

Osmotic shock treatment. An osmotic shock procedure similar to that described by Neu and Heppel (31) was used. Cells were washed twice with 30 mM Tris (pH 8.0) and resuspended to a density of 5 mg (dry weight) of cells · ml¹ in 30 mM Tris (pH 8.0) containing 1 M sucrose and 10 mM EDTA. Following 15 min of incubation at room temperature, cells were collected by centrifugation for 4 min at 14,000 × g, and then periplasmic proteins were released by resuspending the pellet in 0.1 mM MgSO₄ at room temperature. The shock-treated cells were collected by centrifugation, gently resuspended in MMNH₄-OP, and then used for P_i transport assays.

EDTA treatment of cells. Like previous investigators (23, 25, 48), we found that it was necessary to use a mild EDTA treatment to permeabilize the outer membrane in order to use the ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the p probe tetraphenylphosphonium bromide (TPP⁺). P_i cells were washed twice with 30 mM Tris (pH 8.0) and resuspended to a density of 5 mg (dry weight) of cells · ml¹ in 30 mM Tris, and then 1 mM EDTA (pH 7.0) was added. After 5 min of incubation at room temperature, the cells were centrifuged for 4 min at 14,000 × g, washed twice with 30 mM Tris, and resuspended in MMNH₄-OP to a density of 0.5 mg (dry weight) of cells · ml¹. Alkaline phosphatase was not released by this procedure (data not shown), which indicated that the EDTA treatment did not result in release of periplasmic proteins.

Energy coupling. (i) Qualitative determination of membrane potential. CCCP was used to dissipate all components of the p (23, 24). p probes, such as TPP⁺, are passively distributed between the cell and the medium depending on the membrane potential (25) and can be used to assess the effect of CCCP on p (23, 25, 35). TPP⁺ uptake was measured with and without CCCP by using the medium and conditions described above for P_i uptake, except that choline chloride was added to a final concentration of 50 mM. Choline chloride reduces binding of TPP⁺ to anionic groups at the cell surface (28) but does not interfere with P_i uptake (data not shown).

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View this table: [in this window] [in a new window]   TABLE 1.   Effect of p dissipation and ATP depletion on Pi transport in R. tropici CIAT899 and CAP45a

(ii) Determination of ATP concentrations. Intracellular ATP concentrations were determined in experiments in which the effects of CCCP and DCCD were examined. To determine ATP concentrations, the reaction mixtures used were identical to the P_i transport assay reaction mixtures, except that no radioisotope was added. After incubation (see below), cellular ATP was extracted as described by Joshi et al. (23). ATP concentrations were determined by using the luciferase assay, measuring light emission with a Turner model TD-20e luminometer, and employing the internal standard technique (47). Each ATP assay mixture contained 0.05 ml of perchloric acid-treated supernatant, 0.1 ml of 10 mM Tris buffer (pH 8.0), and 0.1 ml of luciferase-luciferin (Promega). After luciferase was injected into the sample and light was measured, an ATP standard was added to the same cuvette and the light was measured again. The amount of ATP in the sample was calculated by using the following equation: ATP concentration = [(RU  RB)/(RIS  RU)] × ATP concentration in the standard, where RU is the luminescence value for the sample, RB is the luminescence value for the blank, and RIS is the luminescence value after addition of the internal standard.

Nucleic acid manipulations. The protocols of Sambrook et al. (36) were used for all routine manipulations of plasmid and chromosomal DNA. The Tn5B22 insertion site in the mutant was characterized by selectively subcloning the transposase portion of Tn5B22 (40) containing the gentamicin resistance gene along with the balance of the transposon and flanking chromosomal DNA. The transposon-chromosome junction was then sequenced, and the resulting nucleotide sequence data were used to conduct a BLASTX search to identify a possible match (4, 16). Briefly, total chromosomal DNA was harvested from the mutant, digested with XmaI, and then ligated into pBluescript KS(+) (Stratagene). The ligation mixture was transformed into E. coli DH5 (36), and plasmids from transformants that were resistant to ampicillin and gentamicin were analyzed by restriction analysis to verify that each contained a single cloned fragment. Southern blotting was then used to verify that the cloned fragment was identical to the fragment in the genome of mutant CAP45. The flanking DNA was sequenced by using an ABI Prism BigDye kit (PE Applied Biosystems, Foster City, Calif.) and an ABI model 310 genetic analyzer (PE Applied Biosystems). The primer 5'-CCATGTTAGGAGGTCACATGGAAGT-CAG-3' was used to initiate sequencing from the transposase terminal (40).

	RESULTS

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Isolation of phosphate transport mutant CAP45. Tn5B22 mutagenesis of R. tropici CIAT899 and selection on minimal GP-gentamicin agar resulted in several gentamicin-resistant mutants that were found to be constitutive for expression of alkaline phosphatase. Enzyme assays of periplasmic extracts obtained from these mutants revealed that they had alkaline phosphatase specific activities of about 800 U (1 U = 1 nmol of p-nitrophenylphosphate hydrolyzed · min¹ · mg of protein¹) when +P_i cells were used. Typically, the alkaline phosphatase activity of CIAT899 +P_i cells is approximately 30 U, and the alkaline phosphatase activity of CIAT899 P_i cells is approximately 1,500 U (1). Presumably, constitutive expression of alkaline phosphatase in the mutants was due to a lack of normal repressive regulatory mechanisms. By screening a subset of the mutants for a P_i transport phenotype we identified isolates that had reduced P_i transport rates. Southern blot analysis of chromosomal DNA prepared from the P_i transport mutants verified that Tn5B22 was present, and all of the blot patterns appeared to be identical, suggesting that the insertion sites were very similar or that the mutants were siblings (results not shown). One representative isolate of these transport mutants was selected for further study; this isolate was designated CAP45.

Kinetic parameters of phosphate uptake. Kinetic plots of P_i transport in both +P_i cells and P_i cells of CIAT899 revealed that two separate transport systems were present. Eadie-Hofstee plots of P_i transport in P_i cells of CIAT899 and CAP45 are shown in Fig. 1. As measured at P_i concentrations of 0.1 to 500 µM and calculated from a linear regression analysis, the estimated K_m values for two transport systems differed by approximately 2 orders of magnitude. In addition to being expressed under high-P_i growth conditions, both systems were induced in response to P_i deprivation, as shown by the increases in the V_max values of P_i cells (Table 2).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Eadie-Hofstee plots of initial Pi uptake velocities in R. tropici CIAT899 and CAP45. Symbols: , CIAT899 HATS; , CIAT899 LATS; , CAP45 LATS. Transport rates were determined with Pi cells as described in the text at Pi concentrations between 0.1 and 500 µM. Each point is the mean of values from three independent experiments; for each experiment three replicate values were obtained at each Pi concentration. The standard error for each data point did not exceed 10% of the mean, and the standard errors are not shown to simplify presentation.

Effect of osmotic shock on phosphate uptake. Osmotic shock release of periplasmic proteins was used to determine if either P_i transport system required a periplasmic solute-binding protein to exhibit the maximal transport rate. P_i uptake was dramatically reduced in osmotic shock-treated cells (Fig. 2). As determined with high and low P_i levels that were saturating for either P_i transport system, osmotic shock reduced the P_i transport rates by approximately 80%. This was the case for both strains and suggested that both the HATS and the LATS depend on a P_i-binding protein for maximal P_i translocating activity.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Effect of osmotic shock on the uptake of Pi in Pi cells of R. tropici CIAT899 and CAP45. Pi uptake was determined for both control cells and shocked cells at Pi concentrations of 5 µM and 400 µM. (A) Pi uptake in wild-type strain CIAT899. (B) Pi uptake in mutant CAP45. Symbols: , 5 µM Pi with shocked cells; , 5 µM Pi with control cells; , 400 µM Pi with shocked cells; , 400 µM Pi with control cells. The value for each time point is the mean of values from three independent experiments for CIAT899 or two independent experiments for CAP45. The error bars indicate the standard errors of the means.

Energy coupling to phosphate transport. To assess the roles of p and ATP in energizing P_i transport, CIAT899 and CAP45 were treated with CCCP and DCCD. The protonophore CCCP dissipates the energized membrane and inhibits processes that use the p directly as a source of energy (i.e., secondary transport systems). However, reactions driven directly by phosphate bond energy should be relatively resistant to the action of this compound. Conversely, the ATPase inhibitor DCCD should significantly reduce ATP levels, and thus ATP-dependent transport activity should also be significantly reduced when DCCD is added. On the basis of these criteria, we examined energy coupling to P_i transport in both CIAT899 and CAP45. Under the conditions used in the assays (pH 7.2), neutrophilic bacteria, such as rhizobia, do not generate a significant chemical potential (pH), and therefore the p consists primarily of the electrical membrane component () (25), which in cowpea rhizobia has been shown to be unaffected by changes in pH (20).

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Effect of CCCP on TPP+ uptake and accumulation in R. tropici CIAT899. CCCP (5 µmol · 0.125 mg [dry weight]1 · ml1) was () or was not () added before [3H]TPP+ (18 µM; specific activity, 27.5 µCi · µmol1) was added. In other experiments (), CIAT899 was allowed to take up [3H]TPP+ for 4 min, CCCP was added after 4.5 min, and then sampling commenced at 5 min. Cell samples were taken at the times shown and as described in Materials and Methods. The data show the typical effect of CCCP on [3H]TPP+ uptake and accumulation and are from one of the three independent assays performed.

Exchange and efflux of phosphate. After dilution of preloaded cells with media containing no P_i or with media containing excess unlabeled P_i, the level of radioactivity in CIAT899 remained constant, implying that neither P_i transport system mediated P_i efflux or exchange of internal P_i with external P_i (Fig. 4A and B). Mutant strain CAP45 behaved similarly (Fig. 4C). CIAT899 cells preloaded with 400 µM [³²P]KH₂PO₄ (to evaluate both transport systems) and diluted 100-fold with medium containing no P_i exhibited high levels of phosphate uptake (Fig. 4B). We assume that this resulted from diluted [³²P]KH₂PO₄ in the medium that was still saturating the HATS and theoretically half-saturating the LATS. In contrast, after cells preloaded in the presence of 5 µM [³²P]KH₂PO₄ were diluted 100-fold with medium containing no P_i, neither the HATS in CIAT899 (Fig. 4A) nor the LATS in CAP45 (Fig. 4C) was saturated with respect to the solute substrate, and therefore the cells exhibited very reduced or no uptake activity.

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Absence of Pi exchange and efflux in R. tropici CIAT899 and CAP45. (A) Pi cells of CIAT899 were loaded with 5 µM [32P]KH2PO4 (specific activity, 22.5 µCi · µmol1) for 4 min and then diluted 100-fold with a medium containing 5 µM [32P]KH2PO4. Symbols: , uptake control; , no Pi (efflux); , 25 µM unlabeled Pi (exchange). (B and C) Pi cells of CIAT899 (B) and CAP45 (C) were loaded with 400 µM [32P]KH2PO4 (specific activity, 22.5 µCi · µmol1) for 4 min and then diluted 100-fold with a medium containing 400 µM [32P]KH2PO4. Symbols: , uptake control; , no Pi (efflux); , 2 mM unlabeled Pi (exchange). The results are typical of the results of two experiments in which this response was documented.

Characterization of the transposon insertion site. A sequence analysis of the chromosomal DNA adjacent to the transposase end of Tn5B22 revealed a 151-bp segment immediately adjacent to Tn5B22 that exhibited 48 to 52% identity and 74 to 78% similarity to KdpE of E. coli (50), Clostridium acetobutylicum (45, 46), and Mycobacterium tuberculosis (11). KdpE is the cytoplasmic response regulator that is paired with the sensor kinase KdpD, and together these proteins govern expression of the high-affinity potassium transport system in response to changes in medium osmolarity or to potassium-limiting conditions.

	DISCUSSION

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Kinetic analysis showed that R. tropici CIAT899 has two P_i transport systems whose kinetic properties differ significantly. In contrast, CAP45 had a single P_i transport system that exhibited low affinity for P_i. At the solute substrate concentrations used in our assays, the apparent lack of transport activity via a HATS in this mutant allowed us to characterize the LATS. The kinetic properties of the R. tropici HATS suggest that it is not atypical. Its apparent K_m (0.45 µM) is very similar to the apparent K_m values reported for the HATS of E. coli (30), P. aeruginosa (26), and A. johnsonii (48). While it exhibited a V_max that is appreciably higher than the V_max values measured for the HATS of E. coli (30) and P. aeruginosa (26), it is very similar to the V_max observed for the HATS of A. johnsonii (48). The K_m of the LATS is much higher and indeed is more consistent with the range of values reported for secondary P_i transport systems in these bacteria (26, 48, 53).

Both P_i transport systems have characteristics that are consistent with ABC-type transporters (8). Both are shock sensitive, losing roughly 80% of their transport activity when periplasmic proteins are lost (Fig. 2 and Table 3). In addition, the ATPase inhibitor DCCD (Table 1) eliminated the transport activities of both systems. In contrast, the p dissipator CCCP, which has been shown to strongly inhibit secondary transport systems (7, 14), affected neither system (Table 1). Under the assay conditions used in routine P_i transport experiments, CCCP either significantly reduced TPP⁺ uptake or caused the release of TPP⁺ that had accumulated in response to an intact membrane potential (Fig. 3). Finally, the unidirectional uptake activity, as shown by the lack of apparent efflux and exchange activity observed with both systems (Fig. 4), also indicates that both the HATS and the LATS belong to the traffic ATPase class of solute transport systems. To summarize, the data obtained in this study suggest that R. tropici CIAT899 has two P_i transport systems. These transport systems differ in their affinities for P_i (Table 2) but are otherwise similar. Both are inducible by P_i limitation (Table 2), are shock sensitive (Fig. 2 and Tables 3), and utilize ATP to engage P_i transport (Table 1). The presence of two P_i transport systems in R. tropici is in contrast to the single P_i transport system reported for some rhizobia (41), and the presence of two functional traffic ATPase primary P_i transporters has not been reported for any of the other bacteria studied thus far (26, 34, 48, 54), including S. meliloti (49).

Additional, but indirect, evidence suggesting that at least the HATS of R. tropici is a multicomponent ABC type of solute transport system comes from the complex Pho phenotype of CAP45. In addition to the absence of a HATS, CAP45 also expresses alkaline phosphatase constitutively. These two traits also occur together in E. coli (12, 13, 52) and S. meliloti (5) mutants whose multicomponent HATS are affected. In both of the latter species, an operon arrangement is involved, and the operon typically includes genes coding for a periplasmic solute-binding protein, two integral membrane proteins, and an ATP-binding protein. Mutations in these operons result in a loss of the high affinity P_i transport function and also result in a loss of normal Pho regulation (i.e., constitutive expression of alkaline phosphatase, the marker enzyme for the P_i stress response). Analysis of the transposon insertion site in CAP45 revealed that the interrupted gene is kdpE. In both E. coli and C. acetobutylicum (46, 50), KdpE has been shown to be the cytoplasmic response regulator of a two-component regulatory pair which includes the sensor KdpD. Also in both of these bacteria, genes coding for KdpDE are arranged in an operon and are located immediately adjacent to the kdp operon, which codes for an ABC-type high-affinity K⁺ transport system that is upregulated in response to low potassium concentrations in the medium or to low osmotic conditions (for reviews see references 3 and 38). In order to assess the effect (if any) of the affected region of the chromosome on the Pho and P_i transport phenotypes of CAP45, efforts to clone and fully characterize this region are currently under way and will be the subject of a subsequent report.

Functional duplication has been found previously in R. tropici (22, 32), and indeed reiteration is not uncommon in members of the Rhizobiaceae (18, 37). Therefore, the presence of two functional P_i stress-inducible P_i transport systems in CIAT899 is not without precedent. Two separate ABC type P_i transport operons that exhibit homology to the E. coli pst operon have been identified in the unrelated organism M. tuberculosis (10, 11, 27), and this finding implies that perhaps there are at least two traffic ATPase P_i transporters in mycobacteria (27). To our knowledge, these systems have not been characterized at the physiological level, and therefore it is not known if they are functional or to what extent they differ in their kinetic properties.

As discussed above, in broth culture R. tropici does not express alkaline phosphatase until the medium P_i concentration decreases to approximately 1 µM (1). The high levels of alkaline phosphatase in R. tropici bacteroids (1) suggest that under normal growth conditions the host plant perhaps distributes small amounts of P_i to the bacteroids and that the P_i concentration in the peribacteroid space may be quite low. Under such conditions, the HATS may be important to P_i acquisition by R. tropici bacteroids. Initial studies on the symbiotic properties of the mutant isolated in this study have shown that in situ P_i acquisition by CAP45 bacteroids is reduced during symbiosis and that the symbiotic competence of this mutant is also reduced (9).