INTRODUCTION

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Through a complex set of biochemical communications, bacteria in the family Rhizobiaceae interact with specific legume hosts to produce root structures known as nodules. Numerous bacterial genes are involved in nodule formation and function. Two of the better-studied examples include the nodulation (nod) genes, which are expressed in response to isoflavones secreted by the plant (reviewed in references 14, 28, and 48), and the dct genes, which are required for uptake of dicarboxylic acids (malate and/or succinate), the primary sources of energy for the bacteria during symbiosis (reviewed in references 13, 33, and 51). In different rhizobia, two-component regulatory systems have been shown to be involved in the positive regulation of both of these activities (20, 21, 40). Two-component regulatory protein systems consist of a sensor histidine kinase which is paired with a cognate-response-regulatory protein. The sensor kinase detects a particular environmental or metabolic signal and transduces the signal to the cell via phosphorylation of the regulator protein (11, 50). When phosphorylated, the regulatory protein is then activated to increase the transcription of target genes under the control of the regulatory pair.

In addition to the sensor kinase, some response-regulatory proteins have been shown to be phosphorylated by other, less specific, secondary mechanisms involving low-molecular-weight phosphorylated compounds such as phosphoramidate, carbamylphosphate, and acetyl phosphate (19, 29-31, 55). Of these, the most extensively studied has been acetyl phosphate. Synthesis of acetyl phosphate is controlled directly by the enzymes acetate kinase and phosphotransacetylase (Fig. 1), which are encoded by the genes ackA and pta, respectively. Both reactions are reversible and, depending on the metabolic activities leading to the synthesis of their substrates, can influence the acetyl phosphate pool size. Factors thus far shown to influence the intracellular concentration of acetyl phosphate include the growth phase, carbon source, and temperature (30, 38, 56). Acetyl coenzyme A (acetyl-CoA) is a substrate for the Pta reaction and is a central metabolic intermediate tied directly to all anabolic processes. Fluctuation of acetyl-CoA levels in response to changing growth conditions could play an important role in determining acetyl phosphate concentrations and, thus, indirectly play a role in affecting gene expression via this less-specific phosphotransferase mechanism. Transcriptional control of ackA and pta would also be expected to be potentially important in this regard; however, comparatively little is known about the regulation of these genes (12).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   The acetyl phosphate pathway in relation to the tricarboxylic acid cycle, which is a major metabolic pathway in S. meliloti bacteroids. The acetyl phosphate pathway is shown with thick boldfaced arrows. Abbreviations: TCA, tricarboxylic acid; ME, malic enzyme; Acs, acetyl-CoA synthase; AckA, acetate kinase; Pta, phosphotransacetylase; Pdh, pyruvate dehydrogenase.

The potential for acetyl phosphate to act as a signaling metabolite in the Rhizobium-legume symbiosis has been briefly examined for the nod and dct regulatory systems mentioned above. Loh et al. (27) recently reported that Bradyrhizobium japonicum NodW can be phosphorylated by acetyl phosphate in vitro, but Gu et al. (23) were unable to demonstrate this alternative phosphorylation mechanism for Sinorhizobium meliloti DctD. Preston et al. (37) and Smith et al. (46) examined the activities of both Pta and AckA in B. japonicum bacteroids and found that the levels of both enzymes increased in parallel with nodule nitrogen fixation (acetylene reduction) activity. This implied that both enzymes might contribute significantly to bacteroid carbon metabolism and, depending on the relative rates of each enzyme, might allow for the accumulation of acetyl phosphate to levels that could, in turn, influence gene expression.

In this study, we characterized a locus in S. meliloti that contains the genes coding for Pta and AckA. We also created pta and ackA mutants to establish metabolic blockades that would either impede the synthesis of acetyl phosphate or favor its accumulation. We used these mutants to assess the potential importance of acetyl phosphate acting as a signaling metabolite in the S. meliloti-alfalfa symbiosis. These mutations also allowed us to ask whether carbon metabolism via the Pta-AckA pathway is important to alfalfa bacteroids for normal symbiotic function.

	MATERIALS AND METHODS

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Strains and growth conditions. The strains of S. meliloti and Escherichia coli used in this study are shown in Table 1. S. meliloti wild-type strain 104A14 (47) was the parent strain from which all mutants were derived. The isolation of the S. meliloti ackA::Tn5B22 mutant strain psi25 was performed as previously described (52), and in this study this strain has been renamed RmMSU4. Transposon Tn5B22 is a derivative of Tn5 (44) and carries a promoterless lacZ gene. When Tn5B22 inserts in the correct orientation relative to a nearby promoter, a lacZ transcription fusion construct that can be used to monitor gene expression is created. The genotypes and construction of other mutants are described below.

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Plasmids and mutagenesis. The plasmids used in this study are also shown in Table 1. DNA flanking the transposon cloned from RmMSU4 was used as a probe to identify a hybridizing colony in blots of a S. meliloti cosmid library, the construction of which was previously described (2). The library insert in the hybridizing cosmid p7C9 was subcloned in part or in full into pBluescript KS(+) and pCPP30 for sequencing and complementation experiments, respectively. The S. meliloti phoB gene from strain 104A14 was previously cloned on a 2.3-kb HindIII fragment (2), and for experiments in this study this fragment was subcloned into pUC19 and inactivated by insertion of an  cassette (18) into the unique EcoRV site located within the phoB coding region. This entire construct was linearized with XbaI and subcloned into pJQ200SK⁺ to create pMLS90. A pta mutation was similarly constructed by insertion of the  fragment into the pta gene. A 9-kb SacI fragment containing the S. meliloti pta gene was subcloned from p7C9 into pBluescript KS(+) which had been partially digested with KpnI and ligated to a  fragment released from pRL453 by KpnI digestion. A clone containing an  insertion within pta was identified by restriction mapping, linearized with XbaI, and then ligated to pJQ200SK⁺ to form pMLS101.

Nucleic acid manipulations. The protocols of Sambrook et al. (41) were used for all manipulations of plasmid and chromosomal DNA. In previous work in which we sequenced the DNA flanking the Tn5B22 insert in RmMSU4, it was determined that the transposon was in the correct orientation for reporting ackA transcription (52). Briefly, total chromosomal DNA was harvested, digested with XmaI or SalI, and then ligated into pBluescript KS(+). The ligated construct was transformed into E. coli DH5, and plasmids from transformants resistant to ampicillin and gentamicin were analyzed by restriction analysis to verify that each contained a single cloned fragment. Southern blotting was used to verify that the cloned fragment was identical to that in the genome from RmMSU4. Sequencing of the flanking DNA was done by the dideoxynucleotide method, using a kit purchased from United States Biochemical (Cleveland, Ohio). Primers 5'-AACGACGGGATCCATAAT-3' and 5'-CCATGTTAGGAGGTCACATGGAAGTCAG-3' were used to initiate sequencing from the lacZ and transposase termini, respectively (44). Large-scale sequencing of segments of p7C9 that contained the complementing pta and ackA genes was accomplished with an ABI 377 DNA sequencer (Perkin-Elmer, Norwalk, Conn.), using synthetic primers complementary to nucleotide sequences determined within the cloned fragments. Sequence homology searches were conducted by using the BLAST network service (3), and sequence alignments were done with the GAP program (15).

RNA isolation and primer extension. Total RNA was harvested from cells by the method described by Schmidt-Goff and Federspiel (42). Primer extension and end labeling of the oligonucleotide primer with [³²P]ATP (DuPont-NEN; 3,000 Ci mmol¹, 10 mCi ml¹) were performed on 20 µg of strain 104A14 RNA as described by Ausubel et al. (4), using polynucleotide kinase and avian myeloblastosis virus reverse transcriptase from Promega (Madison, Wis.). The primer sequence was 5'-GAGCGGCAAGGCCGCAATCCCAATTAT-3'. Sequencing ladders employing the same primer were generated by the dideoxy termination method, using Sequenase II T7 polymerase (United States Biochemical) and Sequetide (DuPont-NEN). The primer extension product with the corresponding sequencing ladder was separated on 6% polyacrylamide-7 M urea sequencing gels.

Phosphate stress induction experiments. For phosphate starvation experiments, cells were grown to mid-exponential phase in MMN broth containing 5 mM P_i and antibiotics as required, washed twice with MMN lacking P_i (25°C), resuspended in P_i-free MMN to an A₅₉₅ of 0.2, and then divided into two cultures. No P_i was added to one of the cultures, while the other culture was amended to 5 mM P_i. Both cultures were incubated in a reciprocal shaking water bath at 30°C. After the desired incubation period, a culture sample was taken for measurement of Pta, AckA, or -galactosidase activity.

Enzyme assays. -Galactosidase activity was measured as previously described (52). For measuring phosphotransacetylase and acetate kinase activities, both P_i-grown and non-P_i-grown cells were washed and resuspended in a buffer that contained 50 mM Tris-HCl (pH 7.5) and 10 mM MgCl₂ and then were disrupted by sonication. The sonicate was centrifuged at 10,000 × g for 5 min to pellet debris, and then the resulting supernatant was removed and stored on ice for use in enzyme assays.

Plant growth and inoculation. Axenic alfalfa plants were cultured in Magenta growth boxes (Sigma, St. Louis, Mo.) that were described previously (32), using the sand-alumina plant culture technique (22) to control the P_i supply at levels that were P_i limiting and non-P_i limiting for the plants (2).

Nucleotide sequence accession number. The nucleotide sequence of the orfA-pta-ackA-fabI gene sequence has been submitted to GenBank (accession no. AF095903).

	RESULTS

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Sequence analysis of the S. meliloti ackA locus. Using transposon Tn5B22 as a promoter probe, we previously identified ackA as one of several S. meliloti genes that are up-regulated in response to P_i stress (52). Identification of the interrupted ackA gene in mutant strain RmMSU4 was accomplished by cloning the transposon and flanking DNA from RmMSU4 (previously designated psi25 [52]), sequencing the transposon-chromosome junctions, and then conducting homology searches of major databases (52). To more extensively characterize this locus, we used a fragment of DNA flanking the transposon to probe an S. meliloti cosmid library (2) and obtained cosmid p7C9, which contained ackA. Restriction mapping, Southern blot analysis, and enzyme assays following P_i starvation treatments confirmed that ackA was preceded by a suitable amount of DNA such that the transcriptional control elements for ackA could be studied (results not shown). A variety of hybridizing DNA fragments were subcloned from p7C9 into pBluescript and either partially or completely sequenced.

Operon studies: mutational and complementation analysis of gene expression. The orfA-pta-ackA-fabI gene arrangement, the locations of Tn5B22 and the  cartridge in the mutant strains, and the different plasmid constructs are shown in Fig. 2. Enzyme activity levels observed under P_i-limiting and non-P_i-limiting conditions with different combinations of the mutant strains and plasmid constructs are presented in Table 2. Consistent with the sequence analysis of the Tn5B22 insertion in ackA, S. meliloti RmMSU4 lacked P_i starvation-inducible AckA activity. P_i starvation-inducible AckA activity could be restored to normal levels when this strain contained cosmid p7C9 (results not shown). However, p7C9 DNA fragments contained in pMLS3, pMLS4, pMLS7, or pMLS8 (Fig. 2) did not result in restoration of inducible AckA activity. For pMLS7 and pMLS8, this was apparently due to the absence of the C-terminal 45 amino acids of AckA beyond the SacI site (Fig. 2). When the complete AckA coding region was present in pMLS6 but the upstream sequence was not (Fig. 2), there was a slightly increased basal level of AckA activity that lacked normal inducibility (Table 2) (similar results were obtained for pMLS5 [data not shown]). Strain RmMSU6, containing pta interrupted by the  cassette (Fig. 2), showed no P_i stress-inducible Pta activity (Table 2). Furthermore, the mutated pta in strain RmMSU6 also eliminated P_i stress-inducible AckA activity (Fig. 2; Table 2). RmMSU4 containing pMLS7 (data not shown) or pMLS8 (Table 2) displayed very high levels of Pta activity in response to P_i limitation; presumably this was due to a gene dosage effect. A pta:: mutation was also introduced into RmMSU4 to create the ackA pta double-mutant strain RmMSU5 (Fig. 2), and as would be predicted, it had no P_i stress-inducible Pta or AckA activity (Table 2). The mutated pta in strain RmMSU5 also eliminated the P_i stress-inducible ackA::lacZ reporter activity (Fig. 2; also see Fig. 5 below). Together, these results indicate that pta and ackA are transcribed as an operon from a promoter located upstream of pta.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Operon and complementation analysis of the S. meliloti SacI-NotI fragment containing the orfA-pta-ackA-fabI operon. An abbreviated SacI-NotI fragment is shown at the top, with thick boldfaced arrows delineating the boundaries of each gene shown. The positions of the restriction sites used in subcloning analysis are as shown. Below the fragment, mutant strain numbers with the location of Tn5B22 transposon or the  interposon in the pta and ackA genes are shown. At the bottom, plasmid numbers, along with their corresponding subclone inserts, are shown relative to the relevant restriction sites. At the right, the Pi starvation (P)-inducible activities of the Pta and AckA enzymes (+), or the lack thereof (), as well as reporter enzyme levels (where relevant) are shown for each construct or strain. The inducibility of all plasmid-borne Pta and AckA activities was assessed in an RmMSU6 mutant background.

Promoter analysis and mutational confirmation of PhoB regulation. A P_i stress-inducible transcriptional start site was identified by primer extension analysis using a primer complementary to bp 671 to 697. The 73- to 74-nucleotide extension products identified two strong start sites, 171 or 172 bp upstream of the putative translational start predicted for OrfA (Fig. 3). Sequence 5' of the transcriptional start was found to have significant similarity to PHO box sequences identified for other S. meliloti P_i-sensitive genes (5) (GenBank accession no. M96263). The alignment of this region with other predicted P_i-regulated promoter regions allows the construction of an S. meliloti consensus PHO box (Fig. 4). The resulting consensus sequence shows similarities to that of E. coli (57). In addition to the PHO box, another feature of the promoter region is a less-understood, purine-rich direct repeat with the sequence 5'-AATAAAA-3'. The repeat is separated by 3 bp and is located in the leader sequence beginning 7 bp downstream from the transcriptional start site. An association of this direct repeat with any regulatory function has not yet been determined.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Determination of the transcriptional start sites for the S. meliloti orfA-pta-ackA-fabI operon by primer extension. RNA was purified from cells of wild-type strain 104A14 after incubation under high-phosphate culture conditions (Phos. +) or under phosphate starvation conditions (Phos. ). Lanes G, A, T, and C show the sequence ladders generated with the same primer as that used in the extension reaction, with pMLS7 as a template. Relevant complementary DNA sequence is shown on the right, and the positions of the start sites are indicated with asterisks.

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Suggested structure of a consensus S. meliloti PHO box. The consensus nucleotide sequence is derived from a comparison of single PHO boxes identified in the promoter region of phnG (GenBank accession no. M96263) and the orfA-pta-ackA-fabI operon, as well as the two tandem PHO boxes reported for phoC (5). The E. coli consensus PHO box (57) is shown at the bottom for comparison. Nucleotides matching the S. meliloti consensus sequence for the 10 region and PHO box are underlined.

View larger version (17K): [in this window] [in a new window]   FIG. 5.   Reporter enzyme activity of an ackA::lacZ transcriptional fusion construct in response to phosphate starvation. Cells of each strain were prepared and assayed as described in Materials and Methods. Reporter enzyme levels (in Miller units) for strains RmMSU4 (ackA::Tn5B22) (), RmMSU5 (pta:: ackA::Tn5B22) (), and RmMSU7 (ackA::Tn5B22 phoB::) () are shown. Data points are the averages of data from duplicate cultures from one of three independent experiments documenting these responses. Reporter enzyme levels failed to increase when washed cells of all three strains were resuspended in high-phosphate medium (results not shown to simplify presentation).

Symbiotic phenotype of AckA and Pta mutants. There were no apparent effects of either the single ackA mutation or the pta ackA double mutation on symbiosis. Dry-matter production of plants inoculated with either mutant was not significantly different from that of plants inoculated with the wild-type strain. When averaged across plants inoculated with the wild-type strain, RmMSU4, and RmMSU5, the mean shoot dry weights ± the standard deviations were 11.3 ± 0.8 and 15.2 ± 0.4 mg · plant¹ for P_i-limited and non-P_i-limited plants, respectively. Uninoculated control plants averaged 5.5 ± 0.2 and 5.7 ± 0.3 mg · plant¹, respectively. The numbers of nodules per plant averaged 5.1 ± 0.3 and 7.9 ± 0.2 for P_i-limited and non-P_i-limited plants, respectively, and the nodule fresh weight per plant for P_i-limited plants was 4.8 ± 0.3 mg · plant¹, compared to 7.5 ± 0.3 mg · plant¹ for plants cultured with high P_i levels. As determined by analysis of variance, all differences in plant dry matter and nodulation between P_i-treated and non-P_i-treated inoculated plants were statistically significant.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, the sequence, organization, and regulation of S. meliloti genes coding for phosphotransacetylase and acetate kinase activity were examined. These genes were determined to be located within an operon that includes an unidentified ORF and, based on partial sequence analysis, likely also contains fabI. The order of these genes in the operon was determined to be orfA-pta-ackA-fabI. A close physical association of pta with ackA and their cotranscription have also been observed in C. acetobutylicum (9), E. coli (24), and M. thermophila (45). Inclusion of the unknown orfA in the operon is unique to S. meliloti, and the future identification of OrfA's function may offer an important clue for determining the role of this operon in the physiology of the cell in general and in response to P_i limitation in particular.

This operon is sensitive to P_i levels in the medium, being up-regulated in response to P_i limitation. The location of the P_i-sensitive transcriptional start site upstream of orfA was confirmed by combining complementation experiments with mutation and primer extension analyses. Two strong adjacent transcriptional start sites were identified, and sequence analysis of the 35 region revealed the presence of a putative PHO box. In other bacteria, the PHO box serves as a target for the phosphorylated response-regulatory protein PhoB (see reference 57 for a review). When a phoB mutation was introduced into the various strains, increased levels of Pta and AckA enzyme activities and ackA::lacZ reporter activity in response to phosphate limitation were abolished. Taken together, the combined evidence strongly argues that S. meliloti genes coding for enzymes directly involved in the synthesis of acetyl phosphate are in an operon controlled by the Pho regulon in response to P_i availability.

Selective mutation of pta and ackA provided an opportunity to assess whether these particular enzymes are important for S. meliloti during symbiosis. Up-regulation of these genes could potentially influence S. meliloti symbiotic function at two different levels. First, the acetyl phosphate concentration could influence alternative signaling routes and thus affect gene expression during symbiosis. There is now overwhelming evidence showing that dicarboxylic acids are the primary energy source for bacteroids (13, 33, 51), with a large proportion of the malate and succinate being routed to acetyl-CoA via a malic enzyme (Fig. 1) (16, 17) for metabolism through the tricarboxylic acid cycle (13, 33, 51). The acetyl-CoA pool size could therefore be substantial, with the flow of carbon through other pathways, such as the Pta-AckA pathway, being possible. Since S. meliloti bacteroid isolation protocols require a significant amount of time and incubation conditions can have significant effects on intracellular concentrations of acetyl-CoA (53), an important direct precursor of acetyl phosphate, reliable measurement of metabolites such as acetyl phosphate in bacteroids can be problematic. However, we reasoned that by creating metabolic blocks at Pta and/or AckA it should be possible to assess whether acetyl phosphate is potentially an important alternative signaling metabolite during symbiosis. If appreciable acetyl-CoA was channeled through this pathway in S. meliloti bacteroids, then inactivation of Pta would diminish acetyl phosphate synthesis, while a block at AckA should provide for an opportunity for acetyl phosphate pools to accumulate. If these mutations had either effect on acetyl phosphate pool size in bacteroids, there appeared to be no consequence for symbiosis, since neither the ackA mutant nor the pta ackA double mutant was different from the wild-type strain with respect to symbiotic phenotype.

A second level at which mutational analysis of pta and ackA could aid in our understanding of S. meliloti bacteroid physiology concerns the relative importance of acetate as an energy source for bacteroids or as a contributor to the acetyl-CoA pool. B. japonicum bacteroid AckA and Pta levels are correlated with nitrogenase activity during soybean nodule ontogeny (37, 46), and B. japonicum bacteroids are capable of using acetate as an energy source to support nitrogen fixation in vitro (36). Together, these reports suggest these enzymes may be important to carbon metabolism in soybean nodule bacteroids. By contrast, Miller et al. (34) reported that while isolated alfalfa nodule bacteroids could use succinate, fumarate, malate, or oxaloacetate to support nitrogenase activity, acetate was ineffective in this regard. This is not inconsistent with the symbiotic phenotype observed for the mutants studied in our experiments, for which the Nod⁺ Fix⁺ symbiotic phenotype of the S. meliloti ackA and pta-ackA mutants suggests that acetate metabolism is not a critical aspect of alfalfa bacteroid physiology. However, because of potential metabolic overlap between the Pta-AckA pathway and that of acetyl-CoA synthase (Acs) (Fig. 1), an ackA-acs double mutant would be required to completely assess this issue.

It is important to note that the pta mutants constructed in these experiments still retained some Pta activity, as demonstrated by the extract-dependent disappearance of acetyl phosphate. Likewise, based on the acetate- and ATP-dependent production of acetyl phosphate, AckA activity was judged to still be present in all of the mutants. While the levels of both apparent enzyme activities in these mutants were reduced by 80 to 90% (Table 2), the presence of acetyl phosphate-synthesizing and acetyl phosphate-consuming enzyme activities at least suggests that S. meliloti has other Pta and AckA enzymes and that these particular enzymes could be actively expressed during symbiosis. Functional reiteration is not an uncommon feature in bacteria in general, and in the context of the S. meliloti-alfalfa symbiosis, perhaps the best example is the occurrence of two distinct S. meliloti malic enzymes; the NAD-dependent malic enzyme is required for symbiosis, whereas the NADP-dependent malic enzyme is not (16, 17). Under the high-stringency conditions employed in the Southern blot analyses of the various mutants in this study, potential additional pta or ackA genes were not apparent (results not shown).

It is also important to note that the lack of a symbiotic phenotype associated with the mutants of these specific P_i stress-inducible enzymes is consistent with our earlier observations regarding the expression of P_i-sensitive genes in S. meliloti bacteroids. We have previously found that PhoB is not required for an effective symbiosis (2). Also, we have shown that several other genes subject to PhoB control in response to P_i limitation are also not required for symbiotic function and do not appear to be up-regulated in bacteroids, regardless of the P_i nutrition status of the host plant (52). Taken together, our data seem to suggest that S. meliloti bacteroids are not limited for P_i, that alfalfa nodule bacteroids are buffered from P_i deprivation when the host plant experiences P_i limitation, and that expression of genes under the control of the regulatory protein PhoB is not necessary for a normal symbiotic relationship with alfalfa. However, there are inconsistencies to this pattern. The P_i stress-inducible high-affinity P_i transport system encoded by phoCDET in S. meliloti has been reported to be required for normal symbiotic function (5, 10). The phoCDET operon is under the control of PhoB (6, 7), which also negatively regulates the recently identified S. meliloti pit low-affinity P_i transport system (6, 7). In cultured cells of S. meliloti, the Pit transporter is expressed under high-phosphate conditions and repressed under low-phosphate conditions (6, 7). In E. coli, most mutations that inhibit P_i transport through the high-affinity P_i transport system also result in constitutive expression of the Pho regulon (57). Mutations in S. meliloti phoC (the promoter-proximal gene in the phoCDET operon) have the same effect (6) and also result in the repression of the low-affinity Pit system (54). Spontaneous mutations that disconnect PhoB control of pit in the phoC mutant restore normal symbiotic function (10, 35). Therefore, it is possible that the symbiotic phenotype resulting from mutations in S. meliloti phoCDET is actually not due to the lack of P_i uptake through the P_i stress-inducible high-affinity P_i transport system system per se but rather is due to mutations that suppress the normal symbiotic expression of Pit and that coincidentally eliminate the other primary mechanism of P_i uptake. Additional studies involving manipulation of the control of Pho regulon expression and P_i transport may provide important information to help resolve this issue.