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. 2003 Sep;185(17):5240–5247. doi: 10.1128/JB.185.17.5240-5247.2003

Yeast Two-Hybrid Studies on Interaction of Proteins Involved in Regulation of Nitrogen Fixation in the Phototrophic Bacterium Rhodobacter capsulatus

Alice Pawlowski 1, Kai-Uwe Riedel 1, Werner Klipp 1, Petra Dreiskemper 1, Silke Groß 1, Holger Bierhoff 1, Thomas Drepper 1, Bernd Masepohl 1,*
PMCID: PMC181009  PMID: 12923097

Abstract

Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnB-NtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnB-GlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.

Rhodobacter capsulatus is a nonsulfur phototrophic purple bacterium (α subdivision of the proteobacteria) which is able to fix atmospheric dinitrogen via a nif-encoded molybdenum-containing nitrogenase. The organization and regulation of nif genes have been analyzed genetically and biochemically in great detail (for a review, see references 45 and 46). As in many other bacteria, ammonium is the preferred nitrogen source in R. capsulatus, and consequently, the highly energy-demanding N2 fixation process is regulated by ammonium availability at three levels (12, 45). R. capsulatus measures the cellular nitrogen status by a nitrogen regulation (Ntr) system similar to that of enteric bacteria (see below). Under nitrogen-limiting conditions (in the absence of ammonium), NtrC becomes phosphorylated, and in turn, NtrC-P activates transcription of the nifA1 and nifA2 genes (level 1), which encode two almost identical nif-specific transcriptional activators. Both NifA proteins are able to activate expression of the structural genes for nitrogenase, nifHDK, and all the other nif genes, resulting in synthesis of nitrogenase. In addition to ammonium control of transcription of nifA1 and nifA2, ammonium leads to inhibition of the activity of both NifA1 and NifA2 (level 2) and to inactivation of nitrogenase (level 3).

The enteric Ntr system comprises five gene products, GlnD (uridylyltransferase/uridylyl-removing enzyme), two trimeric PII signal transduction proteins, GlnB and GlnK, and the two-component regulatory system NtrB/NtrC (for a review, see reference 52). In response to the cellular glutamine/2-ketoglutarate ratio, GlnB and GlnK are regulated by reversible GlnD-mediated uridylylation. When cells are grown under N-limiting conditions, the PII proteins are uridylylated and thus are unable to interact with the sensor kinase NtrB. Under these conditions, NtrB autophosphorylates and subsequently promotes the phosphorylation of the response regulator NtrC. NtrC-P in turn activates transcription of its target genes. Since R. capsulatus contains genes homologous to glnD, glnB, glnK, ntrB, and ntrC, and based on genetic and biochemical data, regulatory mechanism similar to those of the enteric Ntr system have been proposed (27, 34, 45).

In enteric bacteria, NtrB, in concert with GlnB or GlnK, promotes the dephosphorylation of NtrC under conditions of N excess (52), and as a result, expression of NtrC-dependent promoters is switched off. In contrast to the situation in enteric bacteria, in R. capsulatus only GlnB is involved in regulating NtrB activity, whereas GlnK does not seem to play a role in the Ntr signal transduction mechanism (level 1 [12]). Although a glnB mutation results in expression of both nifA1 and nifA2 in the presence of ammonium, NifA-mediated nif gene expression is inhibited under these conditions (level 2 [12]). This posttranslational control of NifA activity is completely abolished in a glnB glnK double mutant but not in single glnB or glnK mutants, suggesting that GlnB and GlnK can substitute for each other in mediating ammonium repression of NifA activity. Similarly, posttranslational ammonium control of nitrogenase activity mediated by the DraT/DraG system (level 3) is relieved in the glnB glnK double mutant.

This paper will focus on yeast two-hybrid studies on the interaction of proteins involved in regulation of the synthesis and activity of molybdenum nitrogenase by ammonium. The utility of two-hybrid assays for analysis of the interaction of proteins playing different roles in nitrogen regulation has already been demonstrated for selected protein pairs, including GlnB-NtrB of Escherichia coli and Klebsiella pneumoniae (41, 43), NtrB-NtrC of K. pneumoniae (42), GlnK-NifL of Azotobacter vinelandii (59), and NifL-NifA of A. vinelandii (36).

As described above, the PII-like proteins GlnB and GlnK of R. capsulatus play central roles in ammonium regulation of the synthesis and activity of nitrogenase. In the present study, we used a yeast two-hybrid-based approach to analyze whether GlnB and/or GlnK mediates signal transduction by direct interaction with NtrB, NifA1, NifA2, DraT, and DraG, and we examined the ability of GlnB/GlnK and NifA1/NifA2 to form heteromeric structures. In addition, we identified two new putative proteins that interact with GlnB and GlnK by screening an R. capsulatus genomic library constructed for use in yeast two-hybrid studies.

(Preliminary results on these yeast two-hybrid studies were presented at the 13th International Congress on Nitrogen Fixation in Canada [44].)

MATERIALS AND METHODS

Strains and plasmids.

The bacterial strains, yeast strains, and plasmids used in this study are shown in Table 1.

TABLE 1.

Strains and plasmids used in this study

Strain or plasmid Relevant characteristicsa Source or reference
R. capsulatus B10S Spontaneous Smr mutant of R. capsulatus B10 33
E. coli
    DH5α Host for pUC plasmids 24
    MC1061 Host for pGAD424.C derivatives (R. capsulatus genomic library) 10
S. cerevisiae
    EGY48 URA3 TRP1 HIS3 6op-LEU2 13
    CG1945 trp1-901 leu2-3,112 LYS2::GAL1UAS-GAL1TATA-HIS3 URA3::GAL417-mer(x3)-CYC1TATA-lacZ 14
Plasmids
    pEG202 lexA-DBD HIS3 Ampr 18
    pJG4-5 PGAL1-B42-AD TRP1 Ampr 18
    pSH18-34 URA3 8op-lacZ Ampr 18
    pGBT9.C GAL4-DBD TRP1 Ampr 8
    pGAD424.C GAL4-AD LEU2 Ampr 8
    pSG31 pJG4-5 derivative containing AD-glnB This study
    pSG32 pEG202 derivative containing DBD-glnK This study
    pSG33 pEG202 derivative containing DBD-glnB This study
    pSG34 pJG4-5 derivative containing AD-glnK This study
    pSG39 pJG4-5 derivative containing AD-ntrB This study
    pSG40 pEG202 derivative containing DBD-ntrB This study
    pHB2 pEG202 derivative containing DBD-nifA1 This study
    pHB3 pJG4-5 derivative containing AD-nifA1 This study
    pHB4 pJG4-5 derivative containing AD-nifA2 This study
    pHB5 pEG202 derivative containing DBD-nifA2 This study
    pJW3 pEG202 derivative containing DBD-draT This study
    pJW4 pJG4-5 derivative containing AD-draT This study
    pJW5 pEG202 derivative containing DBD-draG This study
    pJW6 pJG4-5 derivative containing AD-draG This study
    pAP7 pGBT9.C derivative containing DBD-glnB This study
    pAP8 pGBT9.C derivative containing DBD-glnK This study
    pA6.1 pJG4-5 derivative containing AD-ntrB in-frame fusion (1.9-kb R. capsulatus genomic DNA) This study
    pA6.4 pJG4-5 derivative containing AD-ntrB in-frame fusion (1.5-kb R. capsulatus genomic DNA) This study
    pA7.2 pJG4-5 derivative containing AD-ntrB in-frame fusion (1.2-kb R. capsulatus genomic DNA) This study
    pA9.3 pJG4-5 derivative containing AD-ntrB in-frame fusion (1.8-kb R. capsulatus genomic DNA) This study
    p3.10 pJG4-5 derivative containing AD-nifA2 in-frame fusion (1.6-kb R. capsulatus genomic DNA) This study
    p6.7 pJG4-5 derivative containing AD-draT in-frame fusion (1.1-kb R. capsulatus genomic DNA) This study
    p6.13 pJG4-5 derivative containing AD-pcrA in-frame fusion (0.8-kb R. capsulatus genomic DNA) This study
    pC1.1 pJG4-5 derivative containing AD-era in-frame fusion (0.8-kb R. capsulatus genomic DNA) This study
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a

Sm, streptomycin; Amp, ampicillin.

DNA techniques.

DNA isolation, restriction enzyme analysis, and cloning procedures were carried out following standard methods (60). Plasmid DNA from yeast cells was isolated by phenol-chloroform extraction after treatment of cells with glass beads (19). Restriction endonucleases, T4 DNA ligase, T4 DNA polymerase, and T4 polynucleotide kinase were purchased from MBI Fermentas (St. Leon-Rot, Germany); calf intestinal phosphatase was purchased from Amersham Biosciences (Freiburg, Germany). Enzymes were used as suggested in the manufacturers' instructions. The oligonucleotides used for PCR amplification were purchased from Roth (Karlsruhe, Germany).

Construction of hybrid plasmids and R. capsulatus genomic library for use in yeast two-hybrid studies.

The R. capsulatus genes glnB, glnK, ntrB, nifA1, nifA2, draT, and draG were PCR amplified with chromosomal DNA of the wild-type strain B10S. Appropriate oligonucleotides were designed for amplification of full-length genes flanked by restriction sites allowing us to generate in-frame fusions with either the DNA-binding domain (DBD) or the activation domain (AD) encoded by the Escherichia coli/yeast shuttle vectors pEG202 (lexA-DBD), pJG4-5 (B42-AD), and pGBT9.C (GAL4-DBD).

R. capsulatus B10S chromosomal DNA was isolated following the instructions of the blood and cell culture DNA kit (Qiagen, Hilden, Germany). DNA was sonicated to an average size of 2 kb, repaired with T4 DNA polymerase, and phosphorylated with T4 polynucleotide kinase. Vector plasmid pGAD424.C (GAL4-AD) was digested with SmaI prior to dephosphorylation with calf intestinal phosphatase to avoid religation. After ligation of vector and genomic DNA fragments, the ligation mixture was electroporated into E. coli strain MC1061 as described earlier (61). Transformants were suspended in fresh Luria-Bertani (LB) medium and stored as glycerol stock at −80°C. Plasmid DNA from this library was prepared with the plasmid Giga kit (Qiagen, Hilden, Germany).

Yeast two-hybrid analysis of interaction between defined protein partners.

Yeast strain EGY48(pSH18-34) was cotransformed with two hybrid plasmids (a pEG202 derivative carrying a DBD fusion and the HIS3 marker, and a pJG4-5 derivative containing an AD fusion and the TRP1 marker) by the polyethylene glycol-lithium acetate method as described earlier (17, 19). Transformants were grown on defined dropout medium A (lacking uracil, histidine, and tryptophan and containing 2% glucose) at 30°C for 2 to 3 days. For test assays, three independent transformants were grown overnight in dropout medium A, washed twice in dropout medium B (lacking uracil, histidine, and tryptophan and containing 2% galactose and 1% raffinose) and incubated for 4 h at 30°C to induce the GAL1 promoter on plasmid pJG4-5. Cultures were diluted to an optical density at 600 nm of 0.5, and 2-μl aliquots were patched on agar plates based on (i) dropout medium A, (ii) dropout medium B lacking leucine, (iii) dropout medium A with X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), and (iv) dropout medium B with X-Gal. Growth on leucine-deficient plates and formation of blue patches on X-Gal-containing plates within 3 days of incubation at 30°C indicated interaction of the protein pairs.

Quantitation of β-galactosidase activity.

β-Galactosidase activities of yeast strains were determined by the sodium dodecyl sulfate-chloroform method (7, 50). Three independent yeast transformants were grown overnight in 3 ml of dropout medium A at 30°C. Stationary-phase cultures were diluted (1:10) in dropout medium B and grown at 30°C to mid-log phase (optical density at 600 nm, 0.5 to 0.8) before 1-ml aliquots of culture were assayed for β-galactosidase activity as described earlier (7).

RESULTS AND DISCUSSION

Construction of hybrid plasmids for use in yeast two-hybrid studies and test for self-activation.

The PII-like signal transduction proteins GlnB and GlnK play central roles in the ammonium-dependent control of nitrogen fixation in R. capsulatus on at least three different regulatory levels, as demonstrated by genetic studies (12, 45). To further characterize the role of these two proteins, yeast two-hybrid studies based on the LexA system (18) were carried out in order to identify interacting proteins. As a prerequisite for these studies, the entire coding regions for GlnB, GlnK, and other proteins known to be involved in the regulation of nitrogen fixation were cloned into the E. coli/yeast shuttle vector plasmids pEG202 (coding for the DBD of LexA) and pJG4-5 (coding for the B42 AD). The resulting hybrid plasmids carrying either DBD or AD in-frame fusions with glnB, glnK, and other nif-regulatory genes (Table 1) were cotransformed with the relevant “empty” vector plasmid pJG4-5 or pEG202 into yeast reporter strain EGY48(pSH18-34) in order to analyze the ability of the fusion proteins to activate reporter gene expression (so-called self-activation). Strain EGY48(pSH18-34) carries a chromosomal LEU2 reporter gene and an episomal lacZ reporter gene.

Transcriptional activation of these reporter genes was analyzed (i) by growth on leucine-deficient selective dropout plates and (ii) by the formation of blue colonies on plates containing X-Gal. From all fusions tested in this study, only DBD-NtrB mediated significant background transcriptional activity (self-activation), and therefore, with the exception of hybrid plasmid pSG40, all the other plasmids were suitable for yeast two-hybrid studies (see below).

R. capsulatus GlnB but not GlnK interacts with NtrB in the yeast two-hybrid system.

Similar to the situation in many other bacteria, R. capsulatus GlnB mediates signal transduction to NtrB, as demonstrated by genetic studies (12, 45). There are several lines of evidence for direct interaction between GlnB and NtrB in enteric bacteria. For example, in vitro cross-linking studies demonstrate interaction between E. coli GlnB and NtrB (56, 57), and in vivo experiments based on yeast two-hybrid assays suggest interaction between Klebsiella pneumoniae NtrB and GlnB from either K. pneumoniae or E. coli (41, 43). Therefore, it seemed likely that GlnB and NtrB might also interact directly in R. capsulatus. In order to verify this assumption, we carried out yeast two-hybrid studies as shown in Fig. 1.

FIG. 1.

FIG. 1.

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GlnB-NtrB interaction in a yeast two-hybrid assay. Three independent transformants of EGY48(pSH18-34) strains carrying plasmids pSG33 and pJG4-5 (DBD-GlnB × AD), pEG202 and pSG39 (DBD × AD-NtrB), or pSG33 and pSG39 (DBD-GlnB × AD-NtrB) were grown at 30°C for 3 days on defined dropout plates (Materials and Methods). Activation of the LEU2 reporter gene was measured as growth on leucine-deficient (−Leu) plates (A). Activation of the lacZ reporter gene was demonstrated as formation of blue colonies on plates containing X-Gal (B), and accumulation of LacZ was quantified by determination of β-galactosidase activity (C). β-Galactosidase activities are given in Miller units (50), and the values shown are the averages of three independent transformants, each measured in triplicate.

For this purpose, hybrid plasmids pSG33 (DBD-GlnB) and pSG39 (AD-NtrB) were cotransformed into yeast reporter strain EGY48(pSH18-34). Analysis of three independent transformants demonstrated expression of both reporter genes, as shown by growth on leucine-deficient plates (Fig. 1A) and by β-galactosidase-mediated formation of blue colonies on X-Gal-containing plates (Fig. 1B). In contrast to the yeast reporter strain containing both the DBD-GlnB and the AD-NtrB fusions, control strains containing either DBD-GlnB or AD-NtrB did not show significant growth on leucine-deficient plates and did not form blue colonies on X-Gal plates. This observation was corroborated by quantitation of β-galactosidase activities (Fig. 1C). All in all, these data support a direct interaction between R. capsulatus GlnB and NtrB.

In E. coli, K. pneumoniae, and Azorhizobium caulinodans, GlnK can substitute for GlnB in control of NtrB activity (5, 25, 49), and accordingly, in vitro experiments demonstrated direct interaction between E. coli GlnK and NtrB (6). In contrast to the situation in these bacteria, mutational analysis revealed that R. capsulatus GlnK does not substitute for GlnB in regulation of NtrB (12, 45). In line with the genetic data, we could not detect interaction between GlnK and NtrB in yeast two-hybrid studies with reporter strains containing appropriate DBD-GlnK and AD-NtrB fusions. This finding may be explained by differences in the T-loops of PII-like proteins from E. coli, K. pneumoniae, A. caulinodans, and R. capsulatus. A lysine residue conserved at position 40 in the T-loops of E. coli, K. pneumoniae, and A. caulinodans GlnB and GlnK is thought to be required for NtrB-GlnB interaction (30). In R. capsulatus GlnK, however, a serine residue is found at the corresponding position.

R. capsulatus GlnB and GlnK both interact with NifA1 and NifA2.

In diazotrophic organisms, ammonium control of NifA activity is regulated by PII-like proteins in three different ways. (i) Activation of NifA under N-limiting conditions depends strictly on GlnB, as shown for Azospirillum brasilense, Herbaspirillum seropedicae, and Rhodospirillum rubrum (4, 9, 65). (ii) In A. caulinodans and R. capsulatus, neither GlnB nor GlnK is essential for the activation of NifA, but both PII-like proteins are involved in inactivation of NifA in the presence of ammonium (12, 45, 49). (iii) In Azotobacter vinelandii, K. pneumoniae, and Azoarcus sp. strain BH72, a second protein, NifL, inhibits NifA activity in the presence of ammonium. The transduction of the ammonium signal to the NifL-NifA regulatory system involves GlnK (2, 28, 39). Recently, direct protein-protein interaction between A. vinelandii NifL and GlnK has been demonstrated in vitro (38) and in a yeast two-hybrid system (59).

In contrast to other diazotrophic organisms, R. capsulatus contains two NifA proteins, NifA1 and NifA2, which differ only in their 19 N-terminal amino acid residues (26, 54). These NifA proteins can substitute for each other to activate transcription of all the other nif genes under nitrogen-limiting conditions (47). In the presence of ammonium, both NifA proteins are inactivated by a mechanism mediated by GlnB and GlnK, as shown by genetic studies (12, 45). Accordingly, the yeast two-hybrid assays performed in this study suggested direct interaction for the following protein pairs: GlnB-NifA1, GlnB-NifA2, GlnK-NifA1, and GlnK-NifA2 (data not shown and Fig. 2). Therefore, it seems unlikely that control of R. capsulatus NifA activity by GlnB and GlnK requires further transmitter proteins such as NifL in K. pneumoniae.

FIG. 2.

FIG. 2.

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Protein-protein interactions identified by yeast two-hybrid studies. Screening of an R. capsulatus genomic library identified NtrB, NifA2, DraT, and PcrA as proteins that interact with GlnB and Era as a protein that interacts with GlnK. The other interactions shown in this figure were detected by testing defined protein pairs. As described in the text, we did not detect interactions for the protein pairs GlnK-GlnK, GlnK-NtrB, GlnB-DraG, and GlnK-DraG. Proteins known to be involved in the regulation of nitrogen fixation are emphasized by a grey background. Arrows symbolize interacting protein pairs, indicating which protein was fused to the DBD domain (marked by a dot) or to the AD domain (marked by an arrowhead).

R. capsulatus GlnB and GlnK both interact with DraT.

Several diazotrophic bacteria, including Rhodospirillum rubrum and R. capsulatus, regulate nitrogenase activity via the DraT/DraG system in response to ammonium availability (23, 48). Through biochemical and genetic analysis, it has become clear that the activities of DraT and DraG are themselves subject to posttranslational regulation. DraG is active and DraT is inactive under nif-derepressing conditions. Upon addition of ammonium to an N2-fixing culture, DraG becomes inactive and DraT is transiently activated, resulting in modification (ADP-ribosylation) of nitrogenase reductase (whereby the enzyme is inactivated). After ammonium exhaustion, DraG becomes active, whereupon the modifying group is removed and nitrogenase activity is recovered. Recently, an involvement of PII-like proteins in the mechanism for the regulation of DraT and DraG has been demonstrated (32, 40, 64, 65, 66).

Similarly, DraT-dependent ADP-ribosylation of nitrogenase reductase from R. capsulatus is mediated by GlnB and GlnK, as shown by genetic analysis (12, 45). In line with this finding, both GlnB and GlnK were shown to interact with DraT in yeast two-hybrid studies (data not shown and Fig. 2), suggesting that activation of DraT can be mediated directly by either of the two PII-like proteins. In contrast, no interaction between either GlnB or GlnK and DraG was detected in this study. It is worth mentioning that in many bacteria, including the photosynthetic purple bacterium Rhodospirillum rubrum, PII-like proteins are modified by reversible uridylylation in response to nitrogen status (3, 31, 52). In the absence of ammonium, when DraG is active, PII proteins are in the uridylylated form. Since uridylylation does not take place in Saccharomyces cerevisiae, the yeast two-hybrid system will not detect protein-protein interactions that require uridylylation of one partner. Therefore, at present it remains speculative whether the uridylylated form of GlnB and/or GlnK plays a role in control of DraG activity in R. capsulatus.

Formation of homomeric and heteromeric forms of regulatory proteins.

Both GlnB and GlnK from E. coli form homotrimers, as shown by crystallization studies (for a review, see reference 3). Furthermore, E. coli GlnB and GlnK can form heterotrimers in vivo, and it has been proposed that heterotrimers could facilitate fine tuning of the signal transduction cascade (62). Heterotrimer formation was also observed between Synechococcus GlnB and E. coli GlnB or GlnK when the cyanobacterial protein was expressed in E. coli (16). Based on the high degree of conservation between PII-like proteins from different bacteria, one might therefore expect heterotrimer formation to occur in other organisms, including R. capsulatus, which express more than one form of PII concurrently. Indeed, yeast two-hybrid studies based on DBD-GlnB, AD-GlnB, and AD-GlnK fusions confirmed that R. capsulatus GlnB might form either a homomeric form (GlnB-GlnB) or a heteromeric form with GlnK (data not shown and Fig. 2). However, it remains speculative whether GlnB-GlnB and/or GlnB/GlnK interactions in S. cerevisiae were due to the formation of dimeric or trimeric forms. In contrast to the situation for GlnB, we could not identify GlnK-GlnK interactions in S. cerevisiae. This result might be explained by intrinsic properties of the DBD-GlnK fusion hindering interaction with the AD-GlnK protein. Alternatively, low levels of synthesis and/or high protein turnover might prevent accumulation of the DBD-GlnK fusion protein in S. cerevisiae.

NtrC from enteric bacteria and other transcriptional activators are homodimeric proteins that bind to a site with dyad symmetry in DNA (55, 58, 63). Binding of NtrC to DNA promotes the assembly of a functionally active oligomeric complex (58). Based on similar domain structures of NtrC and NifA and footprinting assays, it has been suggested that the active form of NifA is also likely to be an oligomer (21). As mentioned above, R. capsulatus is the only known diazotrophic organism that contains two almost identical nif-specific transcriptional activators, NifA1 and NifA2. In the present study, the two NifA proteins were shown to interact in yeast two-hybrid assays in all combinations tested (data not shown and Fig. 2). These findings strongly suggest that NifA1 and NifA2 may form homodimers as well as heterodimers in R. capsulatus. Similar to the situation discussed for GlnB-GlnK heterotrimers, formation of NifA1-NifA2 heterodimers could facilitate fine tuning of activation of nif gene transcription.

Since expression of nifA1 is higher than that of nifA2 (27), one might speculate that N2-fixing R. capsulatus cells mainly contain NifA1 homodimers and NifA1-NifA2 heterodimers but only a few NifA2 homodimers. As demonstrated by mutational analysis, either NifA1 or NifA2 is sufficient to activate nif gene transcription (47, 54), indicating that both NifA proteins may bind at least as homodimers to DNA. However, at present it remains speculative whether NifA1-NifA2 heterodimers will also bind to DNA and, if binding occurs, whether heterodimers can mediate the formation of the transcriptionally active open complex. Alternatively, the formation of heterodimers unable to bind to DNA may transiently reduce the amount of active NifA protein in the cell.

Construction and screening of an R. capsulatus genomic library for use in yeast two-hybrid assays.

In addition to analyses of interactions between PII-like proteins from R. capsulatus and known nif regulatory proteins, we were interested in identifying novel proteins that interact with GlnB and GlnK. As a prerequisite for these studies, we constructed hybrid plasmids pAP7 (a pGBT9.C derivative encoding DBD-GlnB) and pAP8 (a pGBT9.C derivative encoding DBD-GlnK), which later served as bait plasmids for screening an R. capsulatus genomic library based on a low-copy GAL4 yeast two-hybrid system (7, 8). The hybrid plasmids pAP7 and pAP8 were transformed into the S. cerevisiae reporter strain CG1945, which carries chromosomal HIS3 and lacZ reporter gene fusions, following the transformation protocol in the Yeast Matchmaker GAL4 two-hybrid system 3 (Clonetech, Palo Alto, Calif.). Neither pAP7 (DBD-GlnB) nor pAP8 (DBD-GlnK) mediated expression of the reporter genes, ruling out self-activation.

The above-mentioned R. capsulatus library is based on the E. coli/yeast shuttle vector pGAD424.C, coding for the AD of the Gal4 transcription factor. This DNA library consisted of 2.3 × 106 independent clones with an insert rate of 92%. Since the size of the R. capsulatus genome is about 3.7 × 106 bp (15), the library was expected to contain in-frame fusions between AD and the coding strand of any given R. capsulatus gene statistically every 11 bp, indicating that our library theoretically covers the R. capsulatus genome severalfold.

About 0.5 mg of plasmid DNA (isolated from a pool of E. coli clones carrying the pGAD424.C-based R. capsulatus library) were used to transform S. cerevisiae strain CG1945 containing either plasmid pAP7 (DBD-GlnB) or pAP8 (DBD-GlnK). To calculate the transformation efficiency, a small aliquot of the transformants was plated on medium lacking leucine to select for the presence of pGAD424.C derivatives. The total number of transformants deduced from these experiments, given in Table 2, demonstrated high efficiency of transformation of yeast cells. The remaining transformants were selected on plates lacking histidine but containing 3 mM 3-amino-1,2,4-triazol to suppress growth resulting from background expression of the HIS3 reporter gene. Colonies appearing after 3 to 5 days of incubation were tested for the production of β-galactosidase by filter assay (7). Clones producing a dark blue color within 3 to 24 h were assumed to contain pGAD424.C derivatives coding for potential proteins that interact with either GlnB or GlnK. The pGAD424.C derivatives were isolated from these clones and reintroduced into CG1945 cells containing either pAP7 (DBD-GlnB), pAP8 (DBD-GlnK), or, as a negative control, the “empty” DBD vector pGBT9.C to confirm the interactions and to exclude “false” positives. Hybrid plasmids from “true” positive clones were sequenced, and genes coding for putative interactors were identified by Blast searches in the R. capsulatus genome database (53).

TABLE 2.

Proteins that interact with GlnB and GlnK, identified by screening of an R. capsulatus DNA library for use in yeast two-hybrid studies

Bait No. of transformants of yeast strain CG1945 Prey No. of independent fusions Full-length protein (amino acids) Interacting region (amino acids)
GlnB 4 × 106 NtrB 4 355 1-355 (3×), 112-355 (1×)
NifA2 1 582 9-544
DraT 1 270 5-270
PcrA 1 852 597-852
GlnK 2 × 106 Era 1 303 61-303
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With plasmid pAP7 (DBD-GlnB) as the bait, we identified seven clones (pA6.1, pA6.4, pA7.2, pA9.3, p3.10, p6.7, and p6.13; Table 1) encoding (putative) proteins that interact with GlnB (Table 2, prey). Six of these clones contained hybrid plasmids coding for known proteins that interact with GlnB: NtrB, NifA2, and DraT (see above), clearly demonstrating the ability of this system to identify mostly true-positives. Among these, four independent clones were shown to carry the ntrB gene region fused to the GAL4 activation domain. Three of them (pA6.1, pA7.2, and pA9.3) contained the full-length ntrB coding region. In detail, the insert sizes in plasmids pA6.1, pA7.2, and pA9.3 were 1.9 kb, 1.2 kb, and 1.8 kb, respectively (Table 1), ruling out that these three clones descended from a single ancestor. The fourth clone (pA6.4) encoded a truncated NtrB protein lacking the 111 N-terminal amino acid residues. NtrB is known to have a modular structure consisting of an N-terminal sensor domain, a central domain containing a conserved histidine residue involved in autophosphorylation, and a C-terminal kinase domain. In vitro studies demonstrated that E. coli GlnB interacts with the kinase domain (29, 56), and recently, yeast two-hybrid studies revealed interaction between K. pneumoniae GlnB and an N-terminally truncated NtrB protein (43). Our results confirm these previous findings, showing that the sensor domain of NtrB is dispensable for interaction with GlnB.

The AD-NifA2 fusion protein interacting with GlnB lacked 8 N-terminal and 38 C-terminal amino acid residues (Table 2), suggesting that (at least) the C-terminal DNA-binding domain of NifA is not essential for interaction with GlnB. Since the only AD-DraT fusion protein interacting with GlnB identified in this study consisted of almost the entire DraT protein lacking only four N-terminal amino acid residues, it remains speculative whether the full-length DraT protein is required for GlnB interaction.

In addition to these known proteins that interact with GlnB (see above), we identified PcrA as a new putative interactor (Table 2). R. capsulatus PcrA is predicted to consist of 852 amino acid residues (53), but the 255 C-terminal residues were sufficient for interaction with GlnB. PcrA belongs to superfamily 1 of helicases, including Rep and UvrD from E. coli (22). These helicases play key roles in many cellular processes by promoting the unwinding of DNA. In this context, it is worth mentioning that oxygen appears to prevent the adoption of a DNA conformation that is necessary, directly or indirectly, for nif gene transcription (35). However, it remains speculative whether PcrA influences the structures of nif promoters and thereby regulates the expression of nif genes in R. capsulatus.

With plasmid pAP8 (DBD-GlnK) as the bait, we identified Era as a new putative protein that interacts with GlnK (pC1.1; Tables 1 and 2). R. capsulatus Era is predicted to consist of 303 amino acid residues (53), but the N-terminal 60 amino acid residues were not required for interaction with GlnK. E. coli Era is an essential GTPase belonging to the Ras-like GTPase superfamily (1). Era is involved in many cellular processes, including cell cycle regulation, ribosome assembly, and energy metabolism (20, 37, 51). Era consists of two distinct domains, an N-terminal domain containing four GTP/GDP-binding motifs and a C-terminal domain containing an RNA-binding motif (11, 67). Our data suggest that interaction between Era of R. capsulatus and GlnK requires the C-terminal domain of Era. The finding that GlnK and Era may interact directly with each other is corroborated by genetic studies in Sinorhizobium meliloti, a soil bacterium that fixes nitrogen in symbiosis with its plant partner, alfalfa (H. Berges, personal communication). As found for E. coli, Era seems to be essential for S. meliloti. However, overexpression of era resulted in a symbiosis-defective phenotype, which was suppressed by a mutation in glnK.

Conclusions drawn from interactions of nif regulatory proteins.

As described above, ammonium control of nitrogen fixation is exerted at three levels: (i) NtrB/NtrC-mediated transcriptional activation of nifA1 and nifA2, (ii) posttranslational regulation of activity of the transcriptional activators of all the other nif genes, NifA1, and NifA2, and (iii) DraT/DraG-mediated reversible ADP-ribosylation of nitrogenase reductase. Genetic analyses demonstrated that the PII-like proteins GlnB and GlnK play central roles in transduction of the ammonium signal, with GlnB being involved in control at all three levels and GlnK acting at levels two and three (12, 45). As summarized in Fig. 2, yeast two-hybrid studies confirmed that GlnB (but not GlnK) interacts with NtrB (level 1). Both GlnB and GlnK were shown to interact with either NifA1 or NifA2 (level 2), suggesting that these PII-like proteins are the immediate regulators of NifA activity. Furthermore, both GlnB and GlnK interacted directly with DraT (level 3), suggesting that PII-like proteins are the immediate regulators of DraT activity.

The detection of GlnB-GlnB and GlnB-GlnK interactions in our two-hybrid studies suggests that GlnB may form both homotrimers and heterotrimers in R. capsulatus. Despite the failure to detect a GlnK-GlnK interaction, it seems likely that GlnK, like GlnB, will also form homotrimers in R. capsulatus. In addition, NifA1 and NifA2 were found to interact in S. cerevisiae in every combination tested, and therefore, NifA1 and NifA2 are expected to form both homodimers and heterodimers in R. capsulatus. As discussed above, heterooligomerization is thought to facilitate fine tuning of the signal transduction cascade and transcriptional activation of nif genes.

Last but not least, screening of an R. capsulatus genomic library identified the ATP-dependent helicase PcrA and the Ras-like protein Era as new putative proteins that interact with GlnB and GlnK, respectively. Due to the proposed functions of PcrA and Era in control of DNA conformation and cell cycle regulation, one might speculate that GlnB-PcrA and GlnK-Era interactions reflect integration of the physiological condition of the cell and cell proliferation.

Acknowledgments

S. cerevisiae strains and E. coli/yeast shuttle vectors for yeast two-hybrid studies were generous gifts from R. Brent and P. L. Bartel. We thank T. Rehmann and J. Wiethaus for carrying out initial experiments.

This work was supported by financial grants from Fonds der Chemischen Industrie.

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