Abstract
Rhizobium leguminosarum bv. viciae expresses an uptake hydrogenase in symbiosis with peas (Pisum sativum) but, unlike all other characterized hydrogen-oxidizing bacteria, cannot express it in free-living conditions. The hydrogenase-specific transcriptional activator gene hoxA described in other species was shown to have been inactivated in R. leguminosarum by accumulation of frameshift and deletion mutations. Symbiotic transcription of hydrogenase structural genes hupSL originates from a −24/−12 type promoter (hupSp). A regulatory region located in the −173 to −88 region was essential for promoter activity in R. leguminosarum. Activation of hupSp was observed in Klebsiella pneumoniae and Escherichia coli cells expressing the K. pneumoniae nitrogen fixation regulator NifA, and in E. coli cells expressing R. meliloti NifA. This activation required direct interaction of NifA with the essential −173 to −88 regulatory region. However, no sequences resembling known NifA-binding sites were found in or around this region. NifA-dependent activation was also observed in R. etli bean bacteroids. NifA-dependent hupSp activity in heterologous hosts was also absolutely dependent on the RpoN σ-factor and on integration host factor. Proteins immunologically related to integration host factor were identified in R. leguminosarum. The data suggest that hupSp is structurally and functionally similar to nitrogen fixation promoters. The requirement to coordinate nitrogenase-dependent H2 production and H2 oxidation in nodules might be the reason for the loss of HoxA in R. leguminosarum and the concomitant NifA control of hup gene expression. This evolutionary acquired control would ensure regulated synthesis of uptake hydrogenase in the most common H2-rich environment for rhizobia, the legume nodule.
Hydrogen gas that is evolved during nitrogen fixation in legume root nodules can be recycled through the activity of an H2-uptake (Hup) system synthesized by certain strains of rhizobia. This H2 recycling system, whose first component is a membrane-bound, dimeric, and nickel- and iron-containing hydrogenase, has a potential to increase legume productivity (1, 2). The first report on the isolation of genes encoding the hydrogenase structural subunits in symbiotic bacteria was published in 1983 (3). Since then, a cluster of genes containing, in addition to the structural genes (hupSL), an array of at least 16 genes coding for accessory proteins required for the synthesis of an active hydrogenase has been identified and sequenced in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae (4, 5). Many of these genes are similar to those found in other H2-oxidizing bacteria such as Alcaligenes eutrophus, Rhodobacter capsulatus, Azotobacter vinelandii, and Escherichia coli (6, 7). In R. leguminosarum bv. viciae UPM791, the hydrogenase gene cluster (hupSLCDEFGHIJK hypABFCDEX) is located in the large pSym plasmid, which also contains the nif and fix genes (8).
Considering the complexity of hydrogenase gene clusters and metabolic diversity of the H2-oxidizing microorganisms, multiple factors and regulatory circuits are expected to control hydrogenase gene expression. In free-living B. japonicum cells, induction of hydrogenase activity requires microaerobic conditions, H2, and nickel. These three environmental factors regulate hydrogenase activity at the transcriptional level by acting at the same cis site in the promoter of the hydrogenase structural genes (9). This cis element is located upstream of binding sites for σ54 and integration host factor (IHF) proteins, characteristic of positively regulated −24/−12-type promoters present in many nitrogen fixation genes (10). A strict requirement for RpoN (σ54), but not for IHF, has been demonstrated in B. japonicum (11).
A transcriptional activator, HoxA, that may work in concert with σ54-dependent RNA polymerases in the induction of hydrogenase structural genes has been found in A. eutrophus (12) and B. japonicum (13). Because HoxA is a member of the family of two-component regulatory systems (13), other gene products can be predicted to be involved in environmental signal sensing and transduction. Two genes, hupU and hupV, located upstream of hydrogenase structural genes in B. japonicum (14) and R. capsulatus (15) encode proteins with strong homology to the small and large subunits of the hydrogenase, respectively. A role for hupUV as a sensing complex for nickel levels and other environmental factors (O2 or H2) was proposed. In R. capsulatus the hupUV genes form an operon with hupT, which is involved in repression of hydrogenase synthesis (16).
Induction of hydrogenase structural genes in R. leguminosarum has consistently been detected only in symbiotic cells but not in free-living cells grown under conditions known to induce hydrogenase synthesis in other H2-oxidizing bacteria (17). In situ hybridization assays demonstrated that hup genes are temporally and spatially coexpressed with nif genes in pea nodules (18). In this report, we show that R. leguminosarum bv. viciae contains a defective hoxA gene and that the hupS promoter is activated by NifA proteins from Klebsiella pneumoniae, R. meliloti, and R. etli. Activation occurs through NifA binding to a cis element located between positions −173 and −88. This activation, which requires both a σ54 factor and IHF proteins, indicates that the hydrogenase gene system has become part of the nitrogen fixation regulon in R. leguminosarum.
MATERIALS AND METHODS
Bacterial Strains, Plasmids, and Growth Conditions.
Rhizobium leguminosarum bv. viciae UPM791 (StrR Hup+; ref. 8), R. etli CE3 (provided by L. Girard, Universidad Nacional Autónoma de México), Bradyrhizobium japonicum CB1809 (provided by J. Vanderleyden, John Innes Centre, Norwich, U.K.), and K. pneumoniae UNF122 (provided by M. J. Merrick, Catholic University of Leuven, Belgium) were used as wild-type strains. R. etli Fix− strains CFNX247 (nifAΔ::Sp/Sm; ref. 19) and CFNX249 (nifHa::Sp/Sm nifHbΔ::Km) were a gift of L. Girard. K. pneumoniae strains UNF1789 (ntrC209), UNF2561 (rpoN71), and CK263 (nifA2263; ref. 20) were provided by M. J. Merrick. K. pneumoniae UN1655 (nifE4701; ref. 21) was a gift of G. P. Roberts (University of Wisconsin, Madison). Escherichia coli strains ET8000 and its himD (IHF−) derivative SE1000 have been described (22). References to other plasmids are in figure legends or tables. Growth conditions for Rhizobium and Bradyrhizobium were as previously described (23).
General Procedures.
Plasmid DNA preparation, restriction enzyme digestion, DNA cloning, DNA sequencing, agarose and polyacrylamide gel electrophoresis, and autoradiography were performed by standard methods (24). Serial deletions of plasmid pHL 315 were obtained by exonuclease III–nuclease S1 treatment. The coding probability of nucleotide sequences was analyzed by the method of Gribskov et al. (25) with a R. leguminosarum codon usage table. Immunoblot analysis was carried out as described by Rey et al. (26).
Bacteroid Preparation and Enzyme Assays.
Pea (Pisum sativum L. cv. Frisson) and bean (Phaseolus vulgaris L. cv. Contender) plants were used as hosts for R. leguminosarum bv. viciae and R. etli, respectively. Conditions for plant inoculation and growth, and bacteroid preparation, were as previously described (8). Transcriptional activity was monitored by measuring β-galactosidase activity (27) in cells carrying lacZ gene fusions to the promoters of interest. For E. coli assays, the K. pneumoniae nifA gene encoded by plasmids pMJ220 or pMJ221 was induced with isopropyl-β-d-thiogalactopyranoside (2 mM) for 2 hr before activity determinations. Derepression and assay of nitrogenase activity in K. pneumoniae cultures were carried out as previously described (28).
Purification of His6 Fusion Proteins.
Fusion proteins containing the His6 tag encoded by pRSETb (29)-derived plasmids pHis1 and pHis2 (Fig. 2B) were expressed in E. coli cells using the T7 polymerase/promoter system and [35S]methionine/cysteine labeling (30), and purified by denaturing Ni(II)-nitrilotriacetic-agarose chromatography as described by Rey et al. (26).
RESULTS
Deletion Analysis of the hupSp Regulatory Region.
Symbiotic transcription at the hupS promoter (hupSp) initiates 56 bp upstream of the hupS gene (31). Upstream of the transcription start site, a σ54-binding sequence characteristic of −24/−12-type promoters was identified, suggesting a positive activation of hupSp. To localize potential cis-acting sequences required for symbiotic expression of hupSp, a series of hupSL–lacZ fusions containing sequential 5′ deletions of a DNA region spanning 880 bp upstream of the transcription start site were generated as shown in Fig. 1. Fusion-containing plasmids were transferred into R. leguminosarum UPM791 strain and analyzed for β-galactosidase activity both in aerobically and microaerobically grown vegetative cells and in pea bacteroids (Fig. 1).
β-galactosidase activities associated to plasmid pHL315, which contains the largest promoter fragment, were very low in free-living cells, regardless of the oxygen level. They were comparable to those associated to plasmid pHL54, which has been deleted of all promoter sequences, and were considered as background levels. In contrast, pea bacteroids from strain UPM791 (pHL315) showed a 12-fold increase in their β-galactosidase activity, as compared with that associated to plasmid pHL54. These results indicate a symbiotic requirement for hupSp expression and are consistent with the previous observation of a lack of transcription of hupS in vegetative cells (17). Deletion of 430 (pHL63) and 707 bp (pHL47) from the 5′ end of the insert DNA in plasmid pHL315 had no effect on β-galactosidase activities. However, deletion of a further 85-bp fragment (pHL58) resulted in complete abolishment of symbiotic hupSp expression. This indicates that sequences located downstream of position −173, relative to the transcriptional start site, contain cis-acting elements involved in transcription of hydrogenase genes.
R. leguminosarum bv. viciae UPM791 Contains an Inactive hoxA Locus.
Both in A. eutrophus (6) and B. japonicum (13) hydrogenase gene expression is under the control of a hup-specific transcriptional activator encoded by the hoxA gene. In these organisms, hoxA is located immediately downstream of genes equivalent to R. leguminosarum hypX. Sequence analysis of this region in R. leguminosarum bv. viciae UPM791 revealed the absence of any ORF long enough to encode a HoxA-like protein (Fig. 2A). However, three smaller ORFs encoding amino acid sequences with similarity to different regions of HoxA were identified. The first of them (ORF1, 411 bp), overlaps with hypX by the sequence ATGA, is preceded by a plausible Shine–Dalgarno motif, and exhibited a high coding probability along its whole length. The derived amino acid sequence of ORF1 (136 aa) showed high similarity (60.7% and 57.4% identity) with the N-terminal region of HoxA from B. japonicum and A. eutrophus, respectively (data not shown). Two other ORFs (ORF2, spanning from position 396 to 1253, and ORF3, from position 556 to 1119) were identified downstream of ORF1 (Fig. 2A). These ORFs contained alternating regions of high and low coding probability and are not preceded by plausible Shine–Dalgarno sequences. The derived amino acid sequences from the regions with high coding probability exhibited high similarity (over 40% identity) with the corresponding regions of B. japonicum and A. eutrophus HoxA (data not shown), including the conserved ATP-binding motif GXXGXGKE in ORF2. No other ORFs encoding amino acid sequences with similarity to the C-terminal end of HoxA, including the DNA-binding helix-turn-helix motif (12), were identified downstream of ORF2. These results suggest that frameshift and deletion mutations have accumulated in the hoxA gene, inactivating it. In addition, Southern blot experiments did not reveal the presence of hoxA duplicates in the R. leguminosarum UPM791 genome.
To eliminate the possibility that the frameshifts originate from persistent sequencing artifacts, the actual molecular size of the ORF1 translation product was determined. To this effect, two different DNA fragments spanning the sequenced region (Fig. 2B) were cloned in the His6 vector pRSETb to generate fusion constructs encoding a predicted 114-aa product consisting of a 42 aa His6 N terminus and a 72-aa ORF1 C terminus. A major product of the expected size (ca. 12 kDa) was identified after T7 RNA polymerase-dependent expression and [35S]methionine/cysteine-selective labeling (Fig. 2C, lanes 1 and 2) of both constructs. This product could be purified by Ni(II)-chelate affinity chromatography (Fig. 2C, lanes 4 and 5), thus showing that it contained the His6 tag. Since no translation products larger in size could be identified or isolated, these results show that the pseudo-HoxA protein encoded by R. leguminosarum UPM791 is a truncated, defective product, presumably as a result of the frameshift mutations identified by DNA sequencing.
NifA from Klebsiella pneumoniae and Rhizobium meliloti Activate the hupS Promoter.
Hydrogenase and nitrogenase genes are temporally and spatially coexpressed in pea nodules (18); in addition, hupSp contains RpoN- and IHF-binding motifs characteristic of nif promoters at appropriate distances from the transcription initiation site (Fig. 1B; ref. 31). These results prompted us to investigate whether NifA, the central regulatory component for nitrogen fixation, is involved in symbiotic transcriptional activation of hydrogenase structural genes, despite the fact that no sequences resembling NifA upstream activating sequences (UAS) (TGT-N10-ACA; ref. 35) were found in, or around, the hupSp region absolutely required for symbiotic transcription (Fig. 1B). Experiments with R. leguminosarum nifA mutants were inconclusive because Nif− pea nodules are structurally and developmentally aberrant (18).
The putative NifA-dependence of hupSp expression was tested in the heterologous genetic background of Klebsiella pneumoniae cells under diazotrophic conditions. To this end, plasmid pHL315 was transferred into K. pneumoniae wild-type and ntrC, rpoN, nifA, and nifE mutants, and the β-galactosidase activity of the resulting strains was determined in cells derepressed anaerobically for nitrogenase (Table 1). High levels of β-galactosidase activity were detected in nitrogenase-derepressed K. pneumoniae wild-type and nifE strains, both of which express the nifA gene. In contrast, only background levels of β-galactosidase activity were observed in nifA, ntrC, and rpoN strains, none of which expresses NifA. These results strongly suggest that KpNifA is able to activate the hupSp promoter. The dependence of hupSp promoter activity on the RpoN gene product was tested in K. pneumoniae UNF2561 rpoN (Table 1). Since nifA transcription is also dependent on RpoN, KpNifA was expressed under the control of the lac promoter using plasmid pMJ220 (35). This plasmid was able to restore hupSp activity in the nifA and ntrC mutants, but not in the rpoN mutant (Table 1). This result demonstrates that σ54 is required for NifA-dependent hupSp activity and that NtrC is not implicated in hupSp activation.
Table 1.
Strain* | Genotype | β-Galactosidase, Miller units
|
|||
---|---|---|---|---|---|
Anaerobic | Aerobic | Anaerobic (+pMJ220†) | Aerobic (+pMJ220) | ||
UNF122 | wild type | 964 | 105 | 1,695 | 3,188 |
CK263 | nifA | 69 | 70 | 1,706 | 2,487 |
UNF2561 | rpoN | 24 | 16 | 13 | 17 |
UNF1789 | ntrC | 34 | 2,216 | 3,210 | |
UNF1665 | nifE | 1,125 |
All strains contain plasmid pHL315 (hupSL–lacZ).
pMJ220 expresses the KpNifA protein (35).
It is unlikely that hupSp activation in wild-type K. pneumoniae requires signals resulting from nitrogenase activity (e.g., H2 evolution), since it was also observed in strain UNF1665 (nifE4701), which induces no nitrogenase activity, and in aerobically grown cells containing plasmid pMJ220. However, to exclude indirect effects derived from the nitrogen-fixing system, we tested the proposed NifA-dependent hupSp activation in E. coli, a nondiazotrophic bacterium. Activation of hupSp in E. coli was detected only when KpNifA, encoded by plasmid pMJ220, was present (Table 2). A similar behavior was observed for the NifA-dependent K. pneumoniae and R. meliloti nifH promoters (plasmids pIZ263 and pMB210, respectively), which were included as controls. These experiments were repeated in E. coli cells expressing RmNifA (Table 2). Clear differences in specificity between the two NifA proteins were observed, with RmNifA promoting very poor activation of the K. pneumoniae nifH promoter. However, good hupSp activation was observed in the presence of any of the two NifA proteins. These results again indicate an activation of hupSp by NifA.
Table 2.
Plasmid | (fusion) | β-Galactosidase, Miller units
|
|||
---|---|---|---|---|---|
+RmNifA (pRmW541.10)† | +KpNifA (pMJ220) | +KpNifA* (pMJ221) | −NifA | ||
pHL315 | (hupSL–lacZ) | 2,460 | 1,654 | 47 | 28 |
pHL47 | (hupSL–lacZ) | 1,549 | 1,518 | 71 | 40 |
pHL58 | (hupSL–lacZ) | 195 | 116 | 87 | 45 |
pIZ263 | (nifH–lacZ)‡ | 126 | 5,401 | 3 | 5 |
pMB210 | (nifH–lacZ)§ | 33,635 | 13,423 | 1,030 | 104 |
pRmW541.10 expresses the RmNifA protein (36).
K. pneumoniae nifH–lacZ fusion.
R. meliloti nifH–lacZ fusion.
NifA-Dependent Transcription from hupSp Requires Binding to Upstream cis-Acting DNA Sequences.
The requirement for binding of KpNifA and RmNifA to upstream activating sequences in hupSp for transcriptional activation was investigated by three different approaches. First, activation of hupSp was examined in E. coli cells containing plasmid pMJ221. This plasmid expresses an altered form of the KpNifA protein (KpNifA*) incapable of binding to UAS due to an amino acid substitution (Y512F) in the helix-turn-helix DNA-binding motif at the C terminus of the protein (37). KpNifA* can promote transcription by interacting directly with the RNA polymerase–σ54 complex in those promoters, such as that for nifH in R. meliloti, whose activation is not absolutely dependent on the presence of UAS (38). As shown in Table 2, KpNifA* did not promote transcription from hupSp. In a control experiment, KpNifA* was capable of activating the R. meliloti nifH promoter, but not the K. pneumoniae nifH promoter.
Second, the NifA-dependent activation of hupSp was tested in E. coli cells harboring hupSp deletion derivative plasmids pHL47 and pHL58 (Table 2). As it was the case in R. leguminosarum bacteroids (Fig. 1A), high levels of β-galactosidase activity were associated to plasmid pHL47 but not to plasmid pHL58, thus showing that the same 85-bp upstream DNA fragment, located between positions −173 and −88, is also required for KpNifA- and RmNifA-dependent transcription from hupSp.
Third, the effect of multiple copies of hupSp on K. pneumoniae nitrogenase activity was determined. If NifA activates transcription from hupSp through direct binding, titration of NifA will result in decreased nif gene expression and nitrogenase activity (39). To test this hypothesis, restriction fragments containing the intact or deleted forms of hupSp cloned into the multicopy pBC KS vector were introduced into K. pneumoniae and tested for inhibition of nitrogenase (Table 3). Plasmids pBC315 and pBC47, which contain 880 and 173 bp of DNA upstream of the transcription initiation site, respectively, caused a 90% inhibition of the wild-type level of nitrogenase activity. Meanwhile, plasmid pBC58, which is deleted upstream of position −87, caused no nitrogenase inhibition. This suggests that nitrogen fixation inhibition, and hence NifA binding, is specifically associated with sequences located within positions −173 to −88 of the hupS promoter region.
Table 3.
Strain/plasmid* | hupSp sequences (bp upstream of +1) | Nitrogenase† activity | % inhibition |
---|---|---|---|
UNF122 | None | 0.53 ± 0.15 | 0 |
UNF122 (pBC KS) | None | 0.56 ± 0.18 | 0 |
UNF122 (pBC315) | 880 | 0.06 ± 0.05 | 91 |
UNF122 (pBC47) | 173 | 0.06 ± 0.04 | 90 |
UNF122 (pBC58) | 88 | 0.54 ± 0.10 | 0 |
Inserts from plasmids pHL315, pHL47, and pHL58 were cloned in pBC KS to yield pBC315, pBC47, and pBC58, respectively.
Values [μmol of C2H2 reduced ⋅ h−1 ⋅ (ml of culture)−1] are the average of four independent determinations ± SE.
Symbiotic NifA-Dependent Expression of hupSp.
The NifA requirement for symbiotic expression of hupSp was tested in the R. etli bean system. Contrary to the R. leguminosarum pea system, bean nodules from plants inoculated by Nif− strains were, at least up to the third week postinoculation, structurally similar to Fix+ nodules (data not shown). The hupSp reporter plasmid pHL315 was introduced in the R. etli CE3 wild-type strain, and mutants CFNX247 (nifAΔ::Sp/Sm) and CFNX249 (nifHa::Sp/Sm nifHbΔ::Km), and bacteroid β-galactosidase activities were determined 3 weeks after inoculation. The hupS promoter was expressed in wild-type bacteroids (140 Miller units), but a 6-fold decrease in hupSp expression (24 Miller units) was observed in CFNX247 nifA bacteroids. The normal levels of hupSp activation (147 Miller units) observed with strain CFNX249 (nifHa nifHb) show that the results obtained with the nifA mutant are not due to the Fix− phenotype but specifically to the lack of active NifA.
Integration Host Factor Mediates NifA-Dependent Transcription.
The IHF has been shown to stimulate NifA-mediated activation of nifH transcription by binding to a region located between the NifA UAS and the σ54-binding site (10). Based on sequence similarities, an IHF-binding site was proposed in hupSp (Fig. 1B; ref. 31). The involvement of IHF in NifA-dependent hupSp activation was tested in the E. coli IHF− mutant SE1000 (himD). Only background transcription from hupSp was detected in this strain in the presence of plasmid pMJ220, which constitutively expresses KpNifA (data not shown). A similar behavior was observed with a K. pneumoniae nifH–lacZ fusion construct, whose activation is known to depend on NifA and IHF (10). These results suggest that a R. leguminosarum equivalent to IHF might play a role in hydrogenase gene transcription. However, no such protein has been reported in R. leguminosarum. By using antibodies raised against E. coli IHF, we were able to show the presence of two immunoreactive protein bands of Mr 11 and 13 kDa in cell-free extracts from R. leguminosarum bv. viciae UPM791 pea bacteroids (Fig. 3, lane 3). These proteins were similar in size to the E. coli IHF proteins (Fig. 3, lane 1) and to IHF-like proteins from Bradyrhizobium japonicum (ref. 11; Fig. 3, lane 2), and may correspond to the α and β subunits of the R. leguminosarum IHF complex.
DISCUSSION
The R. leguminosarum bv. viciae uptake hydrogenase system is unique because it is only expressed in symbiosis with the host legume (ref. 17; Fig. 1A). This is in contrast to the situation with other root-nodule bacteria, such as B. japonicum (5) and Azorhizobium caulinodans (40), where the hydrogenase system can be induced under appropriate culture conditions (low pO2, presence of H2 and Ni2+). Expression of R. leguminosarum hydrogenase structural genes (hupSL) in pea bacteroids is controlled from a promoter located upstream of hupS (hupSp), which has the structure of a −24/−12-type, RpoN-dependent promoter (31). Promoters of this type require an enhancer-binding protein for activation (41). Two other lines of evidence support the idea that hupSp is positively regulated. (i) hupSp upstream sequences are required to drive expression of hupSL–lacZ fusions. Promoter deletion experiments showed that these sequences are located within a 85-bp DNA fragment spanning from position −173 to −88 (Fig. 1). This enhancer-like element is expected to be the binding site of the putative activating protein. (ii) Consensus IHF-binding sequences are present between the cis-acting element and the −24/−12 region. IHF proteins are involved in bending the DNA to facilitate the interaction of the activator protein with the RNA polymerase–σ54 complex (10).
Induction of the hydrogenase system under free-living conditions in other bacteria is known to require a hydrogenase-specific response regulator: HoxA in B. japonicum (13) and A. eutrophus (12), and HupR1 in R. capsulatus (42). R. leguminosarum bv. viciae UPM791 was shown to contain a hoxA-like locus, but the hoxA gene has been inactivated by the accumulation of frameshift mutations at the 5′ end and by an insertion or deletion at the 3′ end. As a result, the hoxA-like locus encodes a truncated protein product of only 136 aa residues instead of the 482–484 aa residues found in active HoxA proteins. This truncated protein lacks the key ATP-binding and C-terminal helix-turn-helix DNA-binding motifs. In addition, it is not required for hydrogenase gene expression, because plasmid pAL618, which does not contain the hoxA-like locus, is able to confer regulated hydrogenase activity to naturally Hup− Rhizobium strains (23).
The symbiotic requirement for hydrogenase expression in R. leguminosarum bv. viciae and the structural similarity between hupSp and nif promoters, together with the fact that molecular hydrogen, the substrate of uptake hydrogenase, is an obligate by-product of the nitrogenase reaction (43), suggest that hup genes might be controlled by a symbiotic nitrogen fixation regulatory protein. Results from in situ hybridization experiments with pea nodules (18) support this possibility. In these experiments, a coinduction of nifH/nifA and hupSL genes was observed in a defined cell layer of the nodule interzone II-III. In this interzone, O2 concentration abruptly drops to values below 100 nM (44), and this apparently triggers activation of nif genes. Because NifA is a key regulatory protein in the O2-controlled activation of nif and fix genes (45), it was an obvious candidate for activation of hupSp.
Using heterologous nifA genes from K. pneumoniae (in K. pneumoniae and E. coli) and R. meliloti (in E. coli), NifA-dependent transcriptional activation of hupSp was observed (Tables 1 and 2). This activation required the hupSp upstream DNA sequences necessary for transcription in R. leguminosarum, and did not occur when an altered form of KpNifA, incapable of DNA binding, was used (Table 2). These results rule out an activation mechanism based on direct interaction of NifA with the RNA polymerase–σ54 complex (46) and suggest that NifA activates transcription by binding to enhancing elements in the −173/−88 hupSp region. However, this region did not contain any NifA UAS (5′-TGT-N10-ACA-3′; ref. 35), nor was any present in the ≈4 kb of upstream DNA present in the Hup+ plasmid pAL618 (47). It is well established that most NifA-regulated promoters contain this consensus UAS (45), although NifA activation through binding to promoter sequences differing from the consensus has been recently documented (19). An alternative explanation for the observed NifA-dependent transcriptional activation of hupSp is that, even in the heterologous E. coli host, NifA induces another transcriptional activator, which in turn activates hupSp. This possibility was ruled out by performing multicopy nif inhibition experiments (Table 3). Introduction of multiple copies of hupSp resulted in specific inhibition of nitrogenase. When promoters containing NifA-binding sites are introduced in K. pneumoniae, nif gene expression and nitrogenase activity are inhibited due to titration of NifA. This observation has been used in the past to define sequences important for NifA binding (39, 48) and, in our case, it shows that NifA-mediated activation of hupSp occurs through direct binding to upstream DNA sequences.
Symbiotic expression analyses of hupSp in nifA R. leguminosarum mutants, both by in situ hybridization and by lacZ fusion assays in bacteroids, were inconclusive because Fix− pea nodules are disorganized both in their structure and in their development (18). However, the R. etli bean system was found amenable to this type of analysis. Transcription activation of the R. leguminosarum hupS promoter was observed in R. etli wild-type and nifH bean bacteroids, but not in nifA bean bacteroids.
Two additional observations suggest that hupSp activation by NifA occurs by a mechanism similar to that of well characterized nif promoters (10): (i) Transcription from hupSp was abolished in a rpoN mutant (Table 1), and (ii) IHF was required for NifA-dependent activation in the heterologous host E. coli, and proteins immunologically related to IHF were detected in R. leguminosarum (Fig. 3), suggesting that IHF-mediated DNA bending is important for hupSp activation.
All the above results suggest that NifA binds to sequences in hupSp that differ from the consensus UAS. Identification of these sequences will require direct physical evidence of binding. Recent developments in the establishment of in vitro assays with active purified NifA protein (49) should facilitate this task.
The requirement to coordinate nitrogenase-dependent H2 evolution and H2 oxidation in nodules might be the reason for the NifA control of hupSL expression in R. leguminosarum and the concomitant evolutionary loss of HoxA. B. japonicum, the only other Hup+ root-nodule bacterium studied in depth, is able to grow chemolithotrophically with H2 and CO2 by coordinately expressing ribulose-biphosphate carboxylase and hydrogenase in free-living conditions (50), and requires HoxA for free-living hydrogenase expression (13). In contrast, R. leguminosarum is not able to carry out a chemolithotrophic metabolism and induces hydrogenase activity only in symbiosis. It is possible that the H2-uptake system of R. leguminosarum was originally under the control of a hoxA-like gene and hydrogenase activity was expressed in free-living conditions in a similar way as B. japonicum. However, adaptation to an ecological situation where the capacity for free-living H2 oxidation was no longer advantageous may have led to the loss of a functional hoxA gene and probably also other genes, such as hupT and hupUV, which are potentially involved in signal transduction or in free-living sensing of H2 or Ni. Alternatively, the structural and accessory genes required for synthesis of an uptake hydrogenase system, but not the missing regulatory genes, may have been recently acquired by R. leguminosarum as a cluster through lateral transfer, and maintained in the symbiotic plasmid. In any case, symbiotic control of hup expression through NifA would ensure regulated synthesis of uptake hydrogenase in the most common H2-rich environment for rhizobia, the legume nodule.
Acknowledgments
We are indebted to Mike Merrick, Gary Roberts, Lourdes Girard, Enrique Morett, and Eduardo Santero for strains and plasmids, and to Howard Nash for the generous gift of purified IHF and IHF antiserum. This work was supported by Grants BIO93-0046 from Comision Interministerial de Ciencia y Tecnologia (Spain), CT960027 from European Community Biotech (both to T.R.A.), and PB91-0120 (to J.I.) from Dirección General de Investigación Cientifica y Ténica (Spain).
ABBREVIATIONS
- IHF
integration host factor
- UAS
upstream activating sequences
- KpNifA and RmNifA
NifA proteins from Klebsiella pneumoniae and Rhizobium meliloti, respectively
Footnotes
References
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