INTRODUCTION

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Biological nitrogen fixation is catalyzed by the nitrogenase complex, which consists of two proteins: dinitrogenase (also referred to as MoFe protein) and dinitrogenase reductase (also referred to as Fe protein) (7). Dinitrogenase is an ₂₂ tetramer of the nifDK gene products and contains the active site (FeMo-co) for reduction of N₂, C₂H₂, and other substrates. Dinitrogenase reductase is an ₂ dimer of the nifH gene product and is the unique electron donor to dinitrogenase. This biological process is very energy demanding and is thus tightly controlled.

In all studied nitrogen-fixing bacteria, nitrogen fixation is regulated at the transcriptional level. Transcriptional regulation has been best studied in Klebsiella pneumoniae, a free-living nitrogen-fixing bacterium, and it involves the general nitrogen regulation (ntr) system (47). This ntr regulatory system has also been intensively studied in Escherichia coli (52), and it has been successfully reconstituted in vitro (25-28). This regulatory system involves the products of at least five genes: ntrA, ntrB, ntrC, glnB, and glnD. glnD encodes a bifunctional, uridylyltransferase-uridylyl-removing enzyme (UTase-UR) that is believed to be the sensor of the intracellular concentration of glutamine in the cell. UTase-UR reversibly controls the activity of the P_II protein (the gene product of glnB) by uridylylation or deuridylylation (46, 52). P_II was recently found to be responsible for the sensing of -ketoglutarate (-KG) in E. coli (32), and its roles are described below. The products of ntrB and ntrC belong to the family of two-component regulators. NtrB (NRII) is a histidine kinase that phosphorylates NtrC (NRI) under nitrogen-limiting conditions and also can act as a phosphatase to dephosphorylate NtrC under nitrogen-excess conditions. Both kinase and phosphatase activities of NtrB are regulated by P_II in response to the level of -KG in the cell (25). The phosphorylated form of NtrC acts as a transcriptional activator of nifA (encoding an activator for other nif genes), glnA (encoding glutamine synthetase [GS]), and other operons involved in nitrogen assimilation. ntrA (also referred to as rpoN) encodes a specific sigma factor, ⁵⁴. Under limiting fixed nitrogen conditions, GlnD functions as UTase and uridylylates P_II. The uridylylation of P_II can stimulate kinase activity of NtrB to phosphorylate NtrC. The phosphorylated NtrC thus activates nifA expression. NifA then activates other nif operons, including nifHDK. Under nitrogen-excess conditions, the process is reversed and nif expression is repressed.

Besides the transcriptional regulation of nif expression, nitrogen fixation is also regulated at the posttranslational level in many diverse nitrogen-fixing bacteria, but it has been well characterized only in Rhodospirillum rubrum, Azospirillum brasilense, Azospirillum lipoferum, and Rhodobacter capsulatus, in which it involves reversible mono-ADP ribosylation of dinitrogenase reductase (41, 67). Two enzymes perform this reversible reaction. Dinitrogenase reductase ADP-ribosyltransferase (referred to as DRAT, the gene product of draT) carries out the transfer of the ADP-ribose from NAD to the Arg-101 residue of one subunit of the dinitrogenase reductase homodimer, resulting in inactivation of that enzyme. The ADP-ribose group attached to dinitrogenase reductase can be removed by another enzyme, dinitrogenase reductase-activating glycohydrolase (referred to as DRAG, the gene product of draG), thus restoring nitrogenase activity.

The draTG genes have been cloned, sequenced, and physiologically characterized from R. rubrum, A. brasilense, A. lipoferum, and R. capsulatus (16, 22, 38, 42, 45, 68). The DRAT-DRAG system negatively regulates nitrogenase activity in response to exogenous NH₄⁺ or to energy limitation in the form of darkness shifts (in the cases of R. rubrum and R. capsulatus) or anaerobiosis shifts (in A. brasilense) (38, 45, 66, 68).

The regulation of the ADP-ribosylation of dinitrogenase reductase is effected through the posttranslational regulation of both DRAT and DRAG activities (67). Under nitrogen-fixing conditions, DRAT is inactive and DRAG is active. Following a negative stimulus such as exogenous NH₄⁺ or energy depletion, DRAT is activated and DRAG becomes inactive, resulting in the loss of nitrogenase activity and the modification of dinitrogenase reductase. However, DRAT activation is only transient, and it becomes inactive again even in the continued presence of the negative stimulus. After removal of the negative stimulus, DRAG becomes active again, and it then reactivates dinitrogenase reductase by cleavage of the ADP-ribose group. However, detail of the mechanisms for the regulation of DRAT and DRAG activities are still unknown.

Because NH₄⁺ not only represses nif expression but also stimulates the modification of dinitrogenase reductase, it is possible that these two regulatory systems might share some common signal transduction pathways. Unfortunately, the transcriptional regulation of nif expression has not been well studied in R. rubrum. Previous studies showed that NtrBC are not essential for nif expression in R. rubrum (69), indicating that R. rubrum has a different regulatory mechanism for nif expression than that seen in K. pneumoniae. The glnB has been cloned in R. rubrum, and it is cotranscribed with glnA from two different promoters: low-level expression from a putative ⁷⁰ promoter under high NH₄⁺ conditions and high-level expression from a ⁵⁴ promoter under N₂-fixing conditions (29). NtrC enhances the transcription of glnBA from its ⁵⁴ promoter (29). Consistent with this, an ntrBC deletion causes low levels of both GS activity and protein (69). P_II in R. rubrum can be uridylylated in response to the change of nitrogen status in the cell (30). Efforts to create insertion mutations in glnB and glnA of R. rubrum have not been successful (29; Y. Zhang and G. P. Roberts, unpublished data), suggesting that they might be deleterious under the conditions used.

To further study the regulation of nitrogen fixation in R. rubrum, we have constructed and characterized glnB, glnA, and nifA mutants and describe here their relationship to the regulation of nif expression and to the function of the DRAT-DRAG system.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1. Antibiotics were used at the following concentrations (mg/liter): streptomycin (Sm), 100; kanamycin (Km), 12.5; nalidixic acid (Nx), 6; tetracycline (Tc), 1; gentamicin, (Gm) 10; and chloramphenicol (Cm), 5 (for R. rubrum); and ampicillin (Ap), 100; Km, 25; Gm, 5; Cm, 25; and Tc, 12.5 (for E. coli).

Growth conditions and whole-cell nitrogenase activity assay. R. rubrum was grown in rich SMN medium (16) or in minimal (MN) medium containing 1 mg of NH₄Cl per ml (37) or in malate-glutamate (MG) medium (37) for the derepression for nitrogenase. MN was used for N₂-fixing growth, and it is the same as MN, except that the NH₄Cl is omitted. Whole-cell nitrogenase activity assay and darkness-NH₄Cl treatments have been described previously (65).

Site-directed mutagenesis of glnB and glnA. QuikChange method (Stratagene, La Jolla, Calif.) was used according to the manufacturer's instructions to generate P_II-Y51F and GS-Y398F substitutions. pYPZ201-1 was used as the template, and it contains a 2.5-kb EcoRV fragment of R. rubrum glnBA cloned into pUX19, a suicide vector for R. rubrum. The pUX19 plasmid was derived from pUK21 (62) and contains an oriT fragment from pSU202 (57), constructed by D. P. Lies and G. P. Roberts (unpublished data). After mutagenesis, all mutations were confirmed by DNA sequencing. The plasmids containing either mutated glnB1 or mutated glnA2 (encoding P_II-Y51F or GS-Y398F) were named pYPZ212 and pYPZ211, respectively.

Construction of an R. rubrum strain expressing P_II-Y51F. pYZ212, containing mutagenized glnB1 (encoding P_II-Y51F), was transformed into E. coli S17-1 (57) and then conjugated into R. rubrum by the method described previously (38). Sm^r Nx^r Km^r R. rubrum colonies were selected, and a representative isolate was termed UR654. Since UR654 arose from a single crossover event, it is merodiploid for glnB in the chromosome, with both wild-type and mutant alleles. To obtain a strain with only the mutant allele, UR654 was grown in SMN with Nx (20 mg/ml) for more than 80 generations, plated on SMN plates, and Km^s colonies were identified after replica printing. A total of 32 Km^s colonies were found, at a frequency of 0.2%. About 50% of these contained the mutant glnB allele, as based on sequencing of PCR-amplified glnB, and a representative isolate was designated UR659.

Construction of an R. rubrum strain expressing GS-Y398F. pYPZ211, containing glnB⁺ and glnA2 (encoding GS-Y398F), was digested with BamHI and then religated. This resulted in the removal of an approximately 770-bp fragment containing the entire glnB gene and part of glnA (but not that portion with the glnA2 mutation), yielding pYPZ215. pYPZ215 was transformed into E. coli S17-1 and then conjugated into R. rubrum, selecting Sm^r Nx^r Km^r colonies. These transconjugants had only one functional copy of glnA (either wild-type or glnA2), because only a portion of glnA was present on the suicide vector. An isolate containing the desired glnA2 allele was identified by Western blot analysis with the antibody against R. rubrum GS as a strain that no longer displayed the adenylylated subunit of GS on Western blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Approximately 40% of isolates showed no modification of GS, and a representative isolate, expressing GS-Y398F, was designated UR687.

Construction of an in-frame deletion in glnB (glnB3). pYPZ201-1, containing R. rubrum glnBA, was digested with EcoRV and HincII and then religated, resulting in the deletion of most of glnA, yielding pYPZ214. pYPZ214 was then digested with PstI and BglII, which created an ~200-bp deletion within glnB, and then treated with mung bean nuclease, which can create blunt ends of DNA and also can degrade double-stranded DNA ends at high enzyme concentrations. After ligation and transformation with E. coli DH5 (GIBCO BRL, Gaithersburg, Md.), plasmids were isolated and sequenced. Some plasmids were identified in which mung bean nuclease had removed a single base (T) at the double-stranded end, resulting in a 201-bp in-frame deletion of glnB. The plasmid bearing this in-frame deletion was named pYPZ216, transformed into E. coli S17-1, and then conjugated into R. rubrum. Sm^r Nx^r Km^r colonies were selected, so that the entire plasmid was integrated into chromosome of R. rubrum, yielding UR689. As described for the construction of the P_II-Y51F-expressing strain above, UR689 was grown in SMN with Nx (20 mg/ml) for more than 130 generations, and 100 Km^s colonies were obtained. Some of these isolates were screened by PCR amplification, and approximately 50% contained the internal deletion of glnB. A representative isolate, containing glnB3, was designated UR717.

R. rubrum glnB3 draG

R. rubrum glnB3 draG4

Cloning of nifA and construction of a nifA mutant. Three oligonucleotides nifA-P1 (5'-GGAGCTGTTCGGCCACGAGAAGGGCG-3'), nifA-P2 (5'-CAGTTCTCCAGCTCGCGCACGTTGCC-3'), and nifA-P3 (5'-CGGGCGGCCTTGGCCTGCACCCAGCC-3') were designed from three conserved regions of nifA from other N₂-fixing bacteria. The nifA-P1 falls in the central domain, nifA-P2 falls in the interdomain linker region, and nifA-P3 falls near the C terminus (46). These were used as primers to PCR amplify nifA from R. rubrum. Pfu DNA polymerase (Stratagene, La Jolla, Calif.) was used for the PCR reaction under the following conditions: 100 to 200 ng of R. rubrum DNA, 0.4 µM concentrations of each primer, 200 µM concentrations of each deoxynucleoside triphosphates, 5% dimethyl sulfoxide, and 5 U of Pfu DNA polymerase. The reaction was carried out in 50 µl of 1× Pfu DNA polymerase reaction buffer. The cycling of PCR reactions was as follows: 95°C for 45 s for one cycle; 95°C for 45 s, 50°C for 45 s, and 72°C for 3 min for 30 cycles; and a final incubation at 72°C for 10 min. A 450-bp PCR product was seen in reactions with the nifA-P1 and nif-P2 primers, and an 850-bp fragment was seen with the nifA-P1 and nif-P3 primers. These PCR fragments were then cloned into pBSKS() (Stratagene), yielding pYPZ217 and pYPZ218, respectively. The deduced amino acid sequence from pYPZ217 and pYPZ218 showed high similarity to NifA from other N₂-fixing bacteria.

Construction of a nifA::lacZ fusion. A 1.7-kb BamHI-SalI fragment from pYPZ220-4, containing the promoter region of nifA and part of nifA itself, was subcloned into pBSKS(), yielding pYPZ224. The vector provides a KpnI site for the next cloning step. A 500-bp NheI-KpnI fragment from pYPZ224, containing a region 5' of nifA and a small portion of nifA itself, was inserted into the XbaI-KpnI sites of pHRP309, a broad-host-range transcriptional fusion vector (54), yielding pYPZ228. pHRP309 and pYPZ228 were transferred from E. coli into R. rubrum separately by triparental mating as described previously (38, 68) with pRK2013 as helper plasmid (13). Sm^r Nx^r Gm^r colonies were selected, and these plasmids were isolated from these transconjugants to confirm their presence.

Expression of nifA from R. rubrum and K. pneumoniae. The entire nifA region (ca. 3.8 kb) was amplified by PCR from total DNA of R. rubrum wild type and then cloned into pRK404 (12), yielding pYPZ239. Plasmids of pYPZ239 (containing R. rubrum nifA) and pCK3 (containing K. pneumoniae nifA) (35) were transferred into R. rubrum into by triparental mating.

DNA sequencing. DNA sequences were determined with the ABI PRISM DyeTerminator Cycle Sequencing Kit (Perkin-Elmer, Foster City, Calif.). Sequencing data were analyzed with DNASTAR software programs (DNASTAR, Inc., Madison, Wis.).

Other DNA techniques. MasterPure genomic DNA Purification Kit from Epicentre (Madison, Wis.) was used for total DNA isolation from R. rubrum, with a few minor modifications. A total of 0.5 to 1 ml of cells was used for DNA isolation. After isopropanol precipitation, DNA was resuspended in 500 µl of 0.1 M sodium acetate and 0.05 M morpholine propanesulfonate (MOPS; pH 8.0) and then reprecipitated with 2 volumes of ethanol. This step significantly improved the quality of DNA. The modified transformation method was used (23), and other DNA manipulations were performed by standard methods (56).

Protein immunoblotting. A trichloroacetic acid precipitation method was used to extract protein quickly as described previously (66). Low-cross-linker SDS-PAGE (ratio of acrylamide to bisacrylamide of 172 to 1) was used for protein separation to obtain better resolution of the modified versus unmodified subunits, and the modified subunit migrates more slowly. Proteins from SDS-PAGE were electrophoretically transferred onto a nitrocellulose membrane and then immunoblotted with polyclonal antibody against dinitrogenase reductase or GS and visualized by use of enhanced chemiluminescence Western blotting detection reagents (Amersham, Arlington Heights, Ill.) or horseradish peroxidase color detection reagents (Bio-Rad, Richmond, Calif.).

Assay for -galactosidase in liquid culture. R. rubrum cultures were first grown in SMN medium and then inoculated into SMN, MN, or MG media with a 65-fold dilution. SMN-grown cultures were grown aerobically in the dark for 2 days. MN- or MG-grown cultures were anaerobically grown under light for 2 days. Ten microliters of liquid culture was used for the -galactosidase assay by the standard protocol as described previously (51). The reactions were carried out at 30°C for 3 min.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Nitrogenase is poorly expressed when only P_II-Y51F is present. The glnBA has been cloned from R. rubrum, and the entire glnB and a small portion of glnA have been sequenced previously (29). We and others (29) have tried to generate glnB and glnA insertion mutants without success, presumably because the absence of GS is lethal. To further study the function of P_II, a P_II-Y51F variant was constructed as described in Materials and Methods. Because Tyr-51 is conserved in all P_II proteins and is the site of uridylylation of E. coli P_II, this substitution should alter the regulation of P_II activity (always deuridylylated), but it was not expected to be lethal.

P_II-Y51F alteration affects posttranslational regulation of nitrogenase activity in response to NH₄⁺. In R. rubrum, nitrogenase activity is regulated by the reversible ADP-ribosylation of dinitrogenase reductase catalyzed by the DRAT-DRAG regulatory system in response to changes in NH₄⁺ or energy status. To determine if P_II plays any role in the DRAT-DRAG regulatory system, the regulation of nitrogenase activity was compared in UR2 (wild type) and UR659 (P_II-Y51F) in response to NH₄⁺ addition or the removal of light. As seen before with UR2, the addition of 10 mM NH₄⁺ causes a slow decrease in nitrogenase activity, finally reaching a stable plateau of about 20 to 30% of the initial activity (65). However, after treatment with the same concentration of NH₄⁺, UR659 showed a very rapid loss of nitrogenase activity, with a low percentage of residual activity (Fig. 1A). In contrast to the NH₄⁺ results, UR659 showed a slightly slower response to darkness than did UR2 (Fig. 1B). Similar to that of UR2, nitrogenase activity in UR659 recovered completely when the cells were returned to light (Fig. 1B). These results suggest that P_II plays a significant role in the signal transduction to DRAT-DRAG in response to NH₄⁺ but less of a role in response to darkness.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Regulation of nitrogenase activity by NH4+ (A) and darkness (B) in R. rubrum UR2 (wild type) (), UR659 (PII-Y51F) (), and UR687 (GS-Y398F) (). (A) NH4Cl was added at t = 0 to derepressed cultures UR2, UR659, and UR687 to a final concentration of 10 mM. (B) Derepressed cultures of UR2, UR659, and UR687 were shifted to darkness at t = 0, and cells were returned to light at 60 min. At the times indicated, 1-ml portions of cells were withdrawn and assayed for nitrogenase activity anaerobically under illumination conditions for 2 min. Initial nitrogenase activities (100%) in UR2, UR659, and UR687 were about 1,200, 200, and 1,000 nmol of ethylene produced per h per ml of cells, respectively, at an optical density of 1.0 at 600 nm. Each point represents an average of at least three replicate assays, with about 10% deviation.

P_II affects DRAT activity. The activities of DRAG and DRAT are independently regulated in response to NH₄⁺ or darkness (41, 67). To determine which of these proteins was the target of the P_II-Y51F effect seen above, DRAG was eliminated from the cell so that we could monitor DRAT activity by itself. A strain expressing P_II-Y51F and also lacking DRAG (UR686) was constructed and analyzed. UR686 showed a complete absence of nitrogenase activity (Table 2), and Western analysis showed dinitrogenase reductase to be completely modified under nitrogen-fixing conditions (data not shown). Under similar conditions, a strain with wild-type P_II, but lacking DRAG (UR214), has been shown to have nearly normal nitrogenase activity (Table 2), reflecting the tight posttranslational regulation of DRAT activity as reported previously (38). This result indicates that the negative regulation of DRAT activity is partially abolished in UR659 (P_II-Y51F) under nitrogen-fixing conditions.

Characterization of glnB deletion mutant (lacking P_II). Because of the interesting behavior of strains expressing P_II-Y51F, a strain with a nonpolar in-frame deletion within glnB (UR717) was constructed, as described in Materials and Methods. The ability to construct such a mutant argues that previous failures to create an insertion mutant in glnB were due to the polar effect of the insertion onto glnA. In contrast to the results in UR659 (P_II-Y51F), UR717 grew more slowly in MG medium, with less red pigment than UR2 (wild type) (data not shown). Unlike UR659, UR717 contained no nitrogenase activity under nif-derepressing conditions (Table 2), and Western blots revealed a very low accumulation of dinitrogenase reductase and dinitrogenase proteins in UR717 under nif derepression conditions (data not shown). These results indicate that P_II is essential for significant derepression of nitrogenase in R. rubrum.

Characterization of nifA in R. rubrum. The effect of P_II on nif expression has been reported in other N₂-fixing organisms, and it is mediated through NifA, the positive regulator for the expression of the other nif genes (10, 46). P_II can affect NifA in two ways: expression and activity. In K. pneumoniae under NH₄⁺-excess conditions P_II stimulates NtrB to dephosphorylate NtrC, so that nifA expression is repressed. However, in other bacteria, such as A. brasilense, P_II controls NifA at the posttranslational level (1, 40). To further characterize effects of P_II on nifA in R. rubrum, nifA was therefore cloned and sequenced, as described in Materials and Methods, and the map of the nifA region is showed in Fig. 2. The deduced amino acid sequence of NifA is highly similar to NifA from other N₂-fixing bacteria, especially in the central domain and C terminus (Fig. 3). Like other N₂-fixing bacteria, such as A. brasilense, R. capsulatus, Rhizobium meliloti, Rhizobium leguminosarum, and Bradyrhizobium japonicum (14, 21, 40, 44, 50, 59), NifA from R. rubrum also has an interdomain linker between the central domain and the C terminus. Four cysteines were located in this linker, which is thought to be involved in O₂ sensing (14) (Fig. 3).

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Physical map of nifA region from R. rubrum. Plasmids used for cloning and expression of nifA were indicated.

View larger version (84K): [in this window] [in a new window]   FIG. 3.   Alignment of amino acid sequences of NifA from R. rubrum (Rr), A. brasilense (Ab), R. capsulatus (Rc), Rhizobium meliloti (Rm), A. vinelandii (Av), and K. pneumoniae (Kp). Four cysteine residues () located in interdomain linker might involve in sensing O2.

Expression of nifA is not regulated in R. rubrum. We have previously shown that NtrBC is not required for nitrogen fixation in R. rubrum (69), and consistent with this fact, neither a putative NtrC binding site nor a ⁵⁴-dependent promoter was found 5' of nifA. The effects of P_II noted above must therefore occur through another mechanism. To determine if P_II affects nifA expression, a nifA::lacZ fusion plasmid (pYPZ228) was constructed as described in Materials and Methods (Fig. 2). Both pYPZ228 and pHRP309 (vector alone) were transferred into different R. rubrum backgrounds, and -galactosidase activity was monitored under three growth conditions. As shown in Table 3, some background -galactosidase activity was seen in R. rubrum with pHRP309 (vector), and a similar background activity was seen when pHRP309 was transferred into R. capsulatus (54). However, at least a fivefold increase of -galactosidase activity was seen when pYPZ228 (nifA::lacZ) was introduced. A similar -galactosidase activity was found in different background strains, including wild type (UR708), ntrBC mutant (UR709), nifA mutant (UR711), and glnB1 (P_II-Y51F) mutant (UR710), when grown under N₂-fixing conditions (MG medium). A lower -galactosidase activity was found in the glnB deletion mutant (UR722) when grown in MG medium. This low -galactosidase activity might be due to other physiological perturbance of this glnB mutant in MG medium, because -galactosidase activity in the vector control strain (UR721) was also lower than in other control strains (Table 3). NH₄⁺ and O₂ have no significant effects on the nifA expression in R. rubrum, since a similar high -galactosidase activity was seen in all strains when grown anaerobically in MN medium or aerobically in SMN medium (Table 3). The expression of nifA is therefore apparently constitutive in R. rubrum, and the P_II effects on nif expression noted above must be at the level of NifA activity, although the nature of this P_II-NifA interaction is unknown at present.

View this table: [in this window] [in a new window]   TABLE 3.   -Galactosidase activity in R. rubrum strains containing nifA-lacZ fusion (pYPZ228) or vector alone (pHRP309) under different growth conditions

Expression of nifA from R. rubrum and K. pneumoniae in R. rubrum. Because P_II is apparently required for NifA activity in R. rubrum, we reasoned that a heterologous, constitutively expressed K. pneumoniae nifA should restore nitrogenase activity in R. rubrum glnB mutants, since the activity of K. pneumoniae NifA does not require activation by P_II in K. pneumoniae. In order to test this hypothesis, we introduced pCK3, containing K. pneumoniae nifA expressed from a constitutive promoter (35), into various R. rubrum strains. The expression of K. pneumoniae nifA has some effects on nitrogenase activity in a wild-type background, since UR694 has a slightly lower nitrogenase activity than UR2 does (Table 2). However, the expression of K. pneumoniae NifA from pCK3 restored nitrogenase activity in a R. rubrum nifA mutant (UR697) and glnB mutants (UR695 and UR720), although the nitrogenase activity in these strains is slightly lower than that seen in UR694 (wild type with pCK3) (Table 2). These results are consistent with the hypothesis that effects of mutations in glnB on nif expression in R. rubrum occur through their effects on NifA.

Expression of unregulated GS does not affect nif expression or DRAT-DRAG regulation in R. rubrum. NH₄⁺ itself is not the direct signal for the DRAT-DRAG system, since methionine sulfoximine, an inhibitor of GS, can significantly block the NH₄⁺ effect on nitrogenase activity in both A. brasilense and R. rubrum (19, 34). Consistent with this, intracellular glutamine concentration increases rapidly after the addition of NH₄⁺ in both R. rubrum and A. brasilense (17, 33). We therefore wondered if GS plays any role in the signal transduction from NH₄⁺ to DRAT-DRAG system.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   Western immunoblot of GS in UR2 (wild type), UR659(PII-Y51F), UR687 (GS-Y398F), and UR717 (no PII). Samples of GS were from cultures grown in the presence of a high concentration of NH4+ (MN) or in the absence of NH4+ (MG). MG-grown cells were also treated with 10 mM NH4Cl (MN+N) or shifted from light to dark (MN+D) for 60 min. Similar amounts of total protein were loaded on SDS-PAGE gels and immunoblotted with antibody against R. rubrum GS. Arrow M indicates the position of the modified subunit, and arrow U indicates the position of the unmodified subunit.

Mutations of glnB have no apparent effect on glnA expression or GS modification. In E. coli, K. pneumoniae, and other bacteria, P_II not only regulates the expression of glnA but also controls GS activity. Specifically, the unmodified form of P_II, together with glutamine and -ketoglutarate, stimulates the adenylylation of GS (52). To study the effect of glnB mutations on the synthesis and adenylylation of GS in R. rubrum, the level of GS and its modification were monitored in UR659 (P_II-Y51F) and UR717 (no P_II) in different growth conditions. To examine the levels of GS protein accumulated in the cell, similar amounts of total protein were subjected to SDS-PAGE and then immunoblotted with antibody against R. rubrum GS. The level of GS in UR659 (P_II-Y51F) was very similar to that seen in UR2 (wild type) (Fig. 4), and the levels of GS in UR687 (GS-Y398F) and UR717 (no P_II) were slightly lower than that in UR2. These results suggested that P_II has no drastic effect on glnA expression. GS from UR659 and UR717 showed a very similar pattern of modification, as seen in UR2 (lanes 5 to 8 and lane 13 to 16, Fig. 4), indicating that P_II had no significant effect on the regulation of the modification of GS. These results were somewhat surprising and will be discussed below.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

P_II plays a very important role in nitrogen fixation in R. rubrum because a deletion mutant of glnB (UR717) showed little nitrogenase activity. The effect of P_II on the nif expression is through NifA. Unlike the situation in K. pneumoniae, P_II does not regulate nifA expression, and NifA is synthesized under NH₄⁺ and aerobic conditions in R. rubrum. Apparently, NifA exists in either active or inactive forms, and P_II is required for the activation of NifA activity under N₂-fixing conditions. Similar regulation of NifA activity has been seen in A. brasilense and Herbaspirillum seropedicae (1, 40, 58). However, the mechanism for the activation of NifA activity (whether it is direct or indirect) is unknown in all cases. Recent studies seen in A. brasilense and H. seropedicae have shown that the N-terminal domain of NifA has an inhibitory effect on NifA activity, since NifA activity is no longer inhibited by NH₄⁺ when this N-terminal domain is deleted (1, 58). In K. pneumoniae and A. vinelandii, NifA activity can be inhibited by another regulatory nif protein, NifL, probably by means of a direct interaction (46). However, no NifL homologue has been found in R. rubrum or A. brasilense.

A mutant with an altered P_II (UR659, P_II-Y51F) showed a decrease of nif expression and lower nitrogenase activity (about 10 to 20% that of wild type). This indicated that some NifA is still active in this mutant under N₂-fixing conditions. However, expression of R. rubrum nifA from a multicopy plasmid was able to completely restore nitrogenase activity in P_II-Y51F mutant (UR742) but not in a deletion mutant lacking P_II (UR744). These results argue that while P_II (probably its uridylylated form) is essential for NifA activity, NifA can still be activated even in the presence of unmodified P_II. A simple model that P_II-UMP activates NifA activity and P_II inhibits NifA is probably incorrect in R. rubrum, and it is completely unclear if the effect of P_II on NifA activity is direct or indirect.

This altered P_II (P_II-Y51F) also showed some effect on the DRAT regulation. A very rapid loss of nitrogenase activity was seen in UR659 in response to NH₄⁺, while a slow response was seen in UR2 (wild type). Furthermore, substantial DRAT activity was found in UR659 under N₂-fixing conditions, indicating that DRAT is not properly regulated in this mutant. It is unknown if the effect of P_II on DRAT is direct or indirect, nor is it clear if P_II has any effect on the regulation of DRAG activity.

In R. rubrum, GS can be adenylylated in response to NH₄⁺ addition or a shift to darkness. However, mutations in P_II have no significant effect on the adenylylation of GS. Similarly, no differences in the extent of modification of GS were seen between wild type and a glnB mutant of E. coli and A. brasilense (2, 9, 11, 60, 61). A P_II paralog, named GlnK or P_Z, has been identified in E. coli, K. pneumoniae, A. brasilense, Azorhizobium caulinodans, Rhodobacter sphaeroides, and H. seropedicae (3, 11, 24, 48, 49, 55, 61), and it shows high similarity to glnB (24). Either GlnK or P_II can effectively regulate ATase to adenylylate or deadenylylate GS in E. coli and A. caulinodans (49, 60, 61). Recently, GlnK was found to be involved in the regulation of NifA activity by relief of NifL inhibition in K. pneumoniae (20, 24). It is our working hypothesis that glnK also exists in R. rubrum and might be involved in the modification of GS.

Because NH₄⁺ controls nif expression and DRAT-DRAG activities, it is possible that these two separate regulatory systems share some common signal transduction pathways. Indeed, several mutants have been found in A. brasilense and R. sphaeroides that show high nitrogenase activity even in the presence of high concentration of NH₄⁺, indicating a perturbation of both regulatory systems. Some of these mutants in A. brasilense also showed other phenotypes such as low GS activity, a defect in histidine transport, or a low rate of NH₄⁺ uptake (8, 15, 18, 43). Unfortunately, genes linked to these phenotypes have not been identified. Recently, mutants in cbbM with defects in the Calvin-Benson-Bassham pathway have been found in R. sphaeroides and R. rubrum, and these mutants show high nitrogenase activity in the presence of NH₄⁺ (31). Furthermore, the expression of glnB and glnK was also affected in an R. sphaeroides cbbM mutant (55). These results suggest that P_II or/and GlnK might be involved in nif expression and the DRAT-DRAG regulatory system in R. rubrum, and it will be very interesting to characterize the function of GlnK in R. rubrum.

In summary, we report here the functional characterization of several regulatory genes involved in nitrogen fixation in R. rubrum. P_II plays an important role and is involved in the regulation of NifA activity and is apparently involved in the regulation of DRAT-DRAG system. The further characterization of these regulatory factors will provide important information about the regulation of nitrogen fixation in this and related organisms.