Ökologie des Bodens, Botanisches Institut, RWTH-Aachen, Worringerweg 1, 52056 Aachen, Germany
Correspondence
Bert Boesten
boesten{at}rwth-aachen.de
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ABSTRACT |
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INTRODUCTION |
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Homologues of these regulatory genes and their target genes have been identified in many other rhizobia. From regulatory studies to date, it has become clear that, between the Rhizobium species, many differences exist in the interconnectivity between these regulatory genes and their target genes (Fischer, 1994). In Rhizobium leguminosarum bv. viciae VF39, genes encoding FixL and FixK homologues have also been identified (Patschkowski et al., 1996
). The fixL gene is located immediately downstream from fixK, and the genes probably form an operon. The R. leguminosarum FixL protein is remarkable in that it has a C-terminal extension encoding a receiver domain. No gene encoding a FixJ homologue has been identified in R. leguminosarum bv. viciae VF39.
FixK belongs to the FNR/CRP superfamily of transcriptional regulators (Green et al., 2001). In S. meliloti, FixK is required for the activation of transcription of the fixNOQP operons. These genes code for a high-affinity cbbB3-type terminal oxidase and are essential for an efficient symbiosis (Preisig et al., 1993
, 1996
). In R. leguminosarum VF39, two copies of the fixNOQP operon exist. One copy is located on the c plasmid, just upstream of the fixK gene, and transcribed divergently. The second copy is located on the d plasmid. Despite the strong similarities with the S. meliloti FixK protein, which regulates the fixNOQP operons in that micro-organism, the R. leguminosarum VF39 FixK has been found to be only marginally involved in fixNOQP expression. Besides fixK, another gene encoding an FNR/CRP-type transcriptional regulator has been identified in R. leguminosarum bv. viciae VF39 (fnrN, Colonna-Romano et al., 1990
). FnrN is found to be primarily responsible for activating the fixNc and fixNd promoters. The expression of the fixNOQP genes is also highly reduced in a fixL mutant background (Schlüter et al., 1997
). This suggests that, in R. leguminosarum VF39, FixL may be involved in the regulation of FnrN.
In this work, we demonstrate that this is indeed the case. Furthermore, we show that the C-terminal receiver domain of the R. leguminosarum bv. viciae VF39 FixL protein is an intermediate in the phophoryl relay pathway involved in the oxygen-dependent regulation of the fnrN promoter.
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METHODS |
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Manipulations of the fixL gene.
A DNA fragment containing the entire sequence encoding FixL was amplified from VF39 chromosomal DNA by PCR with oligonucleotide primers 5 and 6 (Table 2; Fig. 4a
). A BamHI restriction site just upstream of the ATG start codon and an EcoRI restriction site immediately downstream from the stop codon were incorporated in the oligonucleotide primers. The PCR product was digested with BamHI and EcoRI and cloned into the GST plasmid pGEX-5x-3 (Amersham Pharmacia Biotech). This resulted in plasmid pAS1, which encodes the FixLc fusion protein (Fig. 1
). The BamHIEcoRI fragment from pAS1 was cloned into pUC19 to give pJSC100. Point mutations were introduced in pJSC100 using the GeneEditor in vitro Site Directed Mutagenesis System (Promega) and mutagenic oligonucleotides 7 and 8 (Table 2
; Fig. 4a
). This resulted in the introduction of BamHI restriction sites between the PAS and the haem-binding domains (pJSC102) and between the HK domain and the C-terminal receiver domain (pJSC106). The truncated BamHIEcoRI fragments were then excised from these plasmids and cloned into pGEX-5x-3. The resulting plasmids, pJSG103 and pJSG107, coded for the FixL3 and FixL7 fusion proteins, respectively (Fig. 1
).
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Protein purification and in vitro phosphorylation.
The Bulk Glutathione S-Transferase (GST) Purification Module (Amersham Pharmacia Biotech) was used to purify GSTFixL fusion proteins from E. coli DH5 sonic lysates. Purification was done according to the manufacturer's instructions. The proteins were eluted from the resin with 10 mM reduced glutathione, 50 mM Tris/HCl, pH 8, and dialysed against 100 mM KCl, 0·1 mM EDTA, 0·1 mM DTT, 50 mM Tris/HCl, pH 8, and 50 % (v/v) glycerol. The proteins were stored at 20 °C until needed. Phosphorylation reactions were carried out as described by Tuckerman et al. (2001)
. No particular measures were taken to create anaerobic conditions for the phosphorylation experiments. The levels of phospho-FixL in the dried acrylamide gels were quantified with a Fujifilm FLA-3000 phosphoimager (Fuji Photo Film Co.).
Construction of the fixKL deletion mutant.
A VF39 derivative with a chromosomal deletion encompassing the fixK, fixL and azu genes was constructed. Plasmid pTP95 contains a 6936 bp EcoRIPstI DNA fragment spanning the fixKLNOQ' region from R. leguminosarum VF39. A 2505 bp HinDIIIKpnI fragment was deleted from pTP95. This deletion removed the entire coding region of the fixL gene, part of the C-terminus of fixK and part of the N-terminus of the azu gene. The remaining 4431 bp fragment was excised with BamHI and EcoRI and cloned into the chloramphenicol resistance gene of pAS269. The sacRB genes from pUM24 were introduced into the PstI site. This resulted in pMK97·9. Plasmid pMK97·9 was conjugated from E. coli S17-1 into R. leguminosarum VF39-TP4 (fixL : : GmR) (Patschkowski et al., 1996). Selecting for loss of gentamicin resistance resulted in a strain from which fixL and parts of the fixK and azu genes were deleted. This strain was designated VF39
KL (Fig. 4
).
Complementation of VF39KL.
The 1253 bp SalIHinDIII fragment from pTP95 was cloned into pJP2 restricted with XhoI and HinDIII. This resulted in pMKJ-N, bearing a fixNc : : uidA gene fusion. A single NotI restriction site in this plasmid was removed by fill-in religation. The 2385 bp KpnI to HinDIII fragment from pTP95 containing the entire fixL gene was cloned into pMKJ-N. The resulting plasmid pMKJ38 contained a complete fixL gene and a fixK gene from which a 120 bp HinDIII fragment was missing. Another derivative, pMKJ7, was found to contain a complete fixK gene. This was probably due to a partial HinDIII digest. The KpnI to NotI fragment from pMKG8 was used to replace the 1681 bp KpnINotI fragment of pMKJ38 and pMKJ7. This resulted in pMKJ8 and pMKJ12, respectively.
These pMKJ plasmids could be used to complement the VF39KL strain in trans and study the effect on the fixNc promoter. However, in order to avoid copy-number effects on gene regulation and be able to test other transcriptional gene fusions, we preferred chromosomal integration of the complementing fragments. Therefore, the KpnI to XbaI (which flank the SalI/XhoI hybrid site) fragments were excised from these pMKJ plasmids and transferred into pK18mob. The pK18mob derivatives (pMKK38-7, -8 and -12) were integrated into the VF39
KL genome by homologous recombination. The single crossover (see Fig. 4
) should take place in the region of homology between the SalI site in fixNc and the HinDIII site in fixK. Successful integrations were verified by PCR with the fixNc and fixK2 oligonucleotide primers. Integration of plasmid pMKK7 into the VF39
KL genome resulted in strain VF39
KL-7, which has a wild-type genotype. Integration of pMKK38 resulted in VF39
KL-38, which has a fixK fixL+ genotype. Plasmid pMKK8 and pMKK12 both carry the truncated fixL gene (fixL
C). The resulting genotype of VF39
KL-8, therefore, is fixK fixL
C, and VF39
KL-12 is fixK+ fixL
C.
Culture conditions.
Aerobic conditions were achieved by shaking 100 ml cultures at 200 r.p.m. at 28 °C in 250 ml Duran bottles (Schott). Microaerobic induction of the fnrN promoter was achieved as follows. The cultures were grown aerobically to an OD600 of about 0·4. The undiluted aerobically grown cultures were transferred to 100 ml Duran bottles (100 ml cultures in a total volume of about 136 ml). The bottles were sealed and incubation was continued while shaking at 200 r.p.m. Samples were taken with a syringe through a rubber septum, maintaining the microaerobic conditions.
GUS activity was assayed as described by Prell et al. (2002). GUS activity is expressed as nmol p-nitrophenol (PNP) released per minute and per OD600 (nmol(minxOD600)1).
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RESULTS |
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We observed that the R. leguminosarum and the R. etli CFN42 FixL proteins and the S. meliloti FixL-related protein have an unorthodox H box sequence: HDFNNLL. A typical type-1 HK domain contains the consensus motif HEhRTPh (h=conserved hydrophobic residue; Kim and Forst, 2001). The majority of the Rhizobium FixL proteins have a HELNQPL H box sequence. In particular, the first residue after the conserved histidine, which is usually a glutamate, is replaced by an aspartate, and the fifth residue, which is almost invariably a proline, is replaced by leucine. Furthermore, there are some topological differences, indicated in Fig. 1
, in the spacing between the haem domain and the H box and between the H box and the downstream nucleotide-binding site. The spacing between the N and G1 boxes in the nucleotide-binding region is increased by 1014 amino acids. All proteins from this BLAST search with similar unorthodox HK domains also possessed a covalently linked downstream receiver domain.
FixL is a haem-containing protein with autophosphorylation activity
R. leguminosarum FixL consists of at least four distinct domains (Fig. 1). Unlike the S. meliloti FixL, the R. leguminosarum protein does not feature significant transmembrane segments in its N-terminus. The protein is therefore probably cytoplasmic, rather than membrane-located. A SMART (Schultz et al., 2000
) analysis shows that the R. leguminosarum FixL features another PAS domain at its N-terminus instead (Fig. 1
). A number of truncated derivatives of FixL, lacking one or more of these domains, was constructed. Each FixL protein was fused with GST in order to facilitate its purification. The FixL proteins were overexpressed in E. coli and batch-purified using a glutathioneSepharose matrix. FixLc is the complete FixL protein with GST fused to its N-terminus. A spectrophotometric scan of the purified FixLc protein (600250 nm) featured an adsorption peak at 395 nm (data not shown). This was expected for a haem-containing protein, and indicates that the iron atom in the haem moiety is in the Fe3+ (ferric) form (Gilles-Gonzalez et al., 1994
). Incubating the protein in the presence of 10 mM DTT under a N2 atmosphere resulted in a shift of this absorption peak to 431 nm. This wavelength indicates that the haem iron is in the reduced Fe2+ (ferrous) form. Both forms of the GST : : FixLc protein were tested for in vitro autokinase activity in the presence of [
-32P]ATP (Fig. 2a
). The ferric form of the protein had significant autophosphorylation activity. Maximum levels of 32P incorporation were obtained in about 15 min, at which time about 6 % of the FixLc protein was phosphorylated. This level of phosphorylation of FixL is comparable to the level of phosphorylation of the S. meliloti FixL when incubated in absence of FixJ (Tuckerman et al., 2001
). The Fe2+ form of the protein, which was incubated simultaneously under identical conditions, was much less active. Therefore, all other GST : : FixL derivatives were tested in vitro for autokinase activity in their ferric form.
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The truncated FixL3 protein still had significant autokinase activity (Fig. 3, lanes 1 and 2). This indicated that the N-terminal PAS domain is not essential for in vitro autophosphorylation activity. However, the incorporation of 32P was significantly lower than that of the FixLc protein (not shown).
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FixL8 is a derivative of FixL3 which contains a TGA stop codon between the HK- and the C-terminal receiver domains. As a result, FixL8 lacks both the N-terminal PAS domain and the C-terminal receiver domain. FixL8 had low autokinase activity, despite the fact that the HK domain was not altered in this protein (Fig. 2b). When FixL8 and FixL7 were incubated together, FixL7 became efficiently phosphorylated, whereas virtually no radioactivity was incorporated into FixL8. This indicates that the HK domain of FixL8 is functional and that the phosphoryl signal is transferred rapidly to the receiver domain in FixL7.
FixL is required for microaerobic induction of the fnrN promoter
A transcriptional gene fusion of the fnrN promoter with the uidA (GUS) reporter gene was constructed in pJP2. This plasmid (pJP-R) was used to monitor the expression of the fnrN promoter under various physiological conditions and in different genetic backgrounds. The pJP-R reporter was introduced into the wild-type (VF39) and the fixKL mutant strain (VF39KL). No significant activity of the fnrN promoter was obtained under aerobic conditions, regardless of the strain used (t=0, Fig. 5
). In a standard sealed-bottle experiment, the headspace was about 36 ml. This amount of air allowed the cultures to grow for approximately 6 h until anaerobic conditions were reached. No differences in growth rate were observed between the various strains. In the wild-type background, the fnrN promoter was induced after about 1 h, and GUS activity rapidly increased to a maximum level of about 250 units (Fig. 5
). No induction of the fnrN promoter was observed in the fixKL mutant.
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It is possible that the phosphorelay pathway leads from the histidine in FixL, via a receiver domain in another transcriptional regulator protein, to the fnrN promoter. In that case, the C-terminal receiver domain of FixL may be dispensable. To investigate this possibility, VF39KL was complemented with the truncated fixL
C gene. The presence of pMKK8 (fixK, fixL
C; Fig. 5
) or pMKK12 (fixK+, fixL
C; not shown) failed to restore fnrN expression. These results indicate that the C-terminal receiver domain of the FixL protein does play an important role in the regulation of the fnrN promoter.
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DISCUSSION |
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The R. leguminosarum bv. viciae VF39 FixL protein was tested for in vitro autophosphorylation activity. The purified full-length FixLc protein featured an absorption peak at 395 nm. This indicated that the iron in the haem moiety was in the ferric form. In this form, the protein had significant autokinase and phosphorylation activity. Reducing the protein to the ferrous form by incubating with DTT under a N2 atmosphere had a negative effect on the autokinase activity. At first glance, this contradicts earlier reports that S. meliloti FixL activity is correlated to the electronic spin state of the haem iron (Gilles-Gonzalez et al., 1995). Recently, it has been reported that the Cys301 residue (located 16 amino acids downstream from the conserved histidine in the HK domain) in S. meliloti FixL causes an aberrant inactivation of the ferric form of the protein. A C301A mutation of the protein results in a FixL that is equally active for autokinase and FixJ phosphorylation in the ferric form as well as in the unliganded ferrous form (Akimoto et al., 2003
). The Cys301 residue is not conserved in the R. leguminosarum FixL, nor is it conserved in any of the other FixL-like proteins. Therefore, it is not surprising that FixL is fully active in the ferric form. It is possible that, under our experimental conditions, a significant part of the reduced protein was liganded with O2. Oxygenation of the ligand may account for the observed reduction in autokinase activity of the ferrous form of the protein.
The function of the N-terminal domain in the R. leguminosarum VF39 FixL protein is not known. PAS domains occur in many sensory proteins and are generally involved in signal sensing by means of an associated cofactor (Taylor and Zhulin, 1999). The N-terminal PAS domain is not essential for autokinase activity. This, however, does not exclude a possible regulatory role for FixL activity. Comparison of phosphorylation rates is difficult, due to the short half-life of the 32P isotope. Conditions of purification and storage may also affect the activity of the FixL proteins. The C-terminal receiver domain had no autokinase activity, as expected. The receiver could be phosphorylated efficiently in trans by FixL3 and the full-length protein. This indicates that, in these proteins, intramolecular transfer of the phosphoryl from the histidine residue in the HK domain to the aspartate residue in the receiver domain (i.e. turnover) may take place. Deleting the C-terminal receiver domain from FixL3 resulted in a protein (FixL8) with low autokinase activity. Nevertheless, the capacity to phosphorylate the receiver domain in trans was retained. In the presence of FixL7, virtually all radioactivity ended up in the receiver domain, and no phosphorylation of the FixL8 protein could be observed. This suggests that the histidine residue in the kinase domain is only phosphorylated transiently, and that predominantly the aspartate residue in the receiver domain is phosphorylated. In the case of the S. meliloti FixL and FixJ proteins, it has been observed that the autokinase and transfer reactions are much faster and more efficient when the two proteins are allowed to form a complex before the ATP is added (Tuckerman et al., 2001
, 2003
). In hybrid proteins, both domains are part of the same protein and therefore very likely always to form a complex.
We observed that the R. leguminosarum FixL has an unorthodox H box sequence and a topologically altered nucleotide-binding region. All HK proteins with a similar unorthodox HK domain that emerged from the BLASTP search also had a covalently linked receiver module immediately downstream of the HK domain. This suggests that there is a structural difference between the phosphorelay mechanism in these hybrid proteins and the phosphorelay in two-component systems in which the HK and receiver domains are located on separate proteins. This difference may be reflected in the low level of autophosphorylation of the R. leguminosarum FixLC protein. In hybrid proteins, there is always a receiver domain present to accept the phosphoryl signal. In two-component systems, a receiver domain may not always be in the vicinity, and the phosphorylated histidine may have to be stabilized by surrounding residues.
The R. leguminosarum bv. viciae VF39 FixL protein plays an important role in the regulation of the fnrN promoter. The fnrN promoter is easily induced under microaerobic conditions, and vigorous shaking in unsealed flasks was required to repress the promoter. When oxygen becomes limited, the fnrN : : uidA gene fusion is induced. A significant reduction in promoter activity was observed in the fixKL strain. Wild-type levels of induction were restored when this strain was complemented with a full-length copy of the fixL gene. Complementation of the fixK mutation was not required. This suggests that FixK is not involved in the positive regulation of the fnrN promoter. When the C-terminal receiver domain was deleted from the fixL gene, induction of the fnrN promoter could not be restored. This indicates that the C-terminal receiver domain of FixL is essential for regulation of the fnrN promoter under free-living microaerobic conditions.
It is clear that the FixL C-terminal receiver domain is required for fast induction of the fnrN promoter when oxygen levels become limited. It is, however, not clear how FixL exerts this effect on the fnrN promoter. From its domain structure, it is unlikely that FixL itself interacts with the fnrN promoter. It has been shown that fnrN is positively autoregulated and requires the alternative transcription factor RpoN (Clark et al., 2001). In another strain of R. leguminosarum bv. viciae, UPM791, FnrN positively and negatively regulates its own transcription (Colombo et al., 2000
). In our hands, fnrN shows little or no autoregulation. Furthermore, FixK could also interact with the anaeroboxes in the fnrN promoter region. Although, in this study, FixK was not essential for fnrN activity, it is still possible that this regulator negatively influences fnrN transcription. FixL may, directly or indirectly, connect with any of these transcription factors in order to exert its effect on the fnrN promoter. Furthermore, under different physiological conditions, other regulatory pathways may operate, and additional environmental signals may have an effect on proteins involved in the oxygen regulation of gene expression.
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ACKNOWLEDGEMENTS |
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Received 12 May 2004;
revised 22 July 2004;
accepted 4 August 2004.
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