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1 Biotechnology Research Institute, CAAS, Beijing, PR China
2 Biology College, China Agricultural University, Beijing, PR China
3 Institut des Sciences du Végétal, CNRS UPR-2355, Gif-sur-Yvette, France
Correspondence
Min Lin
linmin57{at}vip.163.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Biochemistry, Cellular and Molecular Biology, the University of Tennessee, Knoxville, TN 37996, USA.
Present address: Département de Microbiologie, Biologie Moléculaire du Gène chez les Extrêmophiles, Institut Pasteur, 75724 Paris Cedex 15, France.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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54-dependent family of bacterial activators (Morett & Segovia, 1993
). The mode of activation of nif gene transcription by NifA appears to be common to a large number of diazotrophs, whereas the regulation of NifA synthesis and activity differs from one organism to another (Merrick, 2004). NifA activity in the gamma subgroup of proteobacteria is controlled by the antiactivator NifL. Indeed, NifA and NifL form an atypical two-component sensor–regulator system, and NifL modulates the activity of NifA by direct protein–protein interaction (Martínez-Argudo et al., 2004). NifA proteins are structurally similar to each other. This enabled the description of NifA as a multidomain protein (Studholme & Dixon, 2003 and references therein). The catalytic domain of NifA, which interacts with the RNA polymerase, shares a high degree of identity with an AAA+-type ATPase. The C-terminal part is the DNA-binding domain and contains an HTH motif. The N-terminal domain carries a GAF domain. The NifL protein resembles histidine kinase protein (HPK), but NifL is not subject to autophosphorylation. The amino-terminal region contains a PAS domain, and it has been proposed that this region may be involved in O2 sensing (Zhulin et al., 1997). The C-terminal region shows similarity to the GHKL superfamily of ATPases.
The nitrogen fixation ability within the genus Pseudomonas has been questioned for a long time (for reviews see Chan et al., 1994; Lalucat et al., 2006). It is now established that several strains of Pseudomonas stutzeri can fix nitrogen (Vermeiren et al., 1999; Rediers et al., 2004). P. stutzeri A1501, isolated from rice, fixes nitrogen in the free-living state, under microaerobic conditions in media devoid of ammonia (You et al., 1991; Lin et al., 2000; Desnoues et al., 2003). A 30 kb DNA region containing the nitrogen fixation (nif and rnf) genes has been previously characterized and the regulatory nifLA region mapped within the main nif cluster. By using different lacZ fusions, it was observed that nifA controlled the expression of other nif (and rnf) operons and that chromosomal nifLA–lacZ fusion expression was strongly reduced in the presence of oxygen and ammonia (Desnoues et al., 2003). As NtrC and RpoN were also found to control nifLA expression (Desnoues et al., 2003), this suggested that the regulation circuitry in P. stutzeri resembled more the situation in Klebsiella pneumoniae than in Azotobacter species, where nifLA expression is not impaired in the presence of ammonia (Blanco et al., 1993), although P. stutzeri is phylogenetically closer to Azotobacter (Rediers et al., 2004) than to Klebsiella. The objective of this work was to further document the role of NifLA in P. stutzeri A1501. In particular, the properties of a nifL mutant strain non-polar on nifA expression as well as the physical interaction between NifL and NifA were investigated.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. P. stutzeri was grown in minimal lactate medium or in LB medium at 30 °C as described by Desnoues et al. (2003). The yeast strain used for the two-hybird analysis was Saccharomyces cerevisiae PJ69-4A harbouring three reporter genes, lacZ, ADE2 and HIS3, under the control of the GAL promoter (James et al., 1996). Yeast cells were grown in YPD rich medium or in SD minimal medium supplemented with appropriate amino acids, at 30 °C, as described in the manual supplied with the Matchmaker Two-Hybrid System (Clontech). Antibiotics were used at the following concentrations (µg ml–1): ampicillin (Amp), 100; kanamycin (Km), 50; tetracycline (Tc), 10.
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. Nitrogenase specific activity is expressed as nmol ethylene min–1 (mg protein)–1. Protein concentrations were determined by the Bio-Rad protein assay (BSA protein standard). Each experiment was repeated at least three times.
Molecular techniques.
Plasmid isolation, genomic DNA extraction, gel electrophoresis, restriction mapping, transformation and molecular cloning, Western blotting and amplification by PCR (Amersham kit) were performed by standard methods (Sambrook & Russell, 2001) or as recommended by the manufacturers of the products used. Restriction enzymes were purchased from Promega and oligonucleotides from Shanghai Biotech Company. Nucleotide sequencing was performed by the Takara Company.
RT-PCR.
Total RNA was isolated by acid-phenol extraction and the single-stranded cDNA synthesis was performed using the ProtoScript First Strand cDNA Synthesis Kit (New England Biolabs). RNA (1 µg) isolated from bacteria grown under nitrogen fixation conditions was used for RT-PCR to amplify a 415 bp fragment with primers specific for nifA, RT-PCRnifAF and RT-PCRnifAR (Table 2). After 35 cycles of PCR amplification (94 °C for 1 min; 58 °C for 1 min; 72 °C for 1 min), PCR products were separated on 0.8 % agarose gels and visualized by ethidium bromide staining.
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), except that it carries the KIXX cassette in the same orientation as the nifA gene. A nifL polar mutant, designated 1507A, was constructed for this work, as follows. Oligonucleotide primers pL-F and pL-R (see Table 2
) were designed to amplify a 1647 bp BamHI–HindIII fragment that was cloned at the unique BamHI–HindIII sites of the pSUP202 vector. Then, the KIXX cassette was inserted, as a SmaI fragment, into the unique ApaI site within the nifL coding sequence, previously treated with the Klenow fragment of DNA polymerase I to fill in ends. The orientation of the Km resistance gene was the opposite of that of the nifA gene. Plasmids were transferred into P. stutzeri recipients by conjugation, using Escherichia coli S17-1 as the donor, as previously described (Desnoues et al., 2003). Recombination in the host genome at the correct location was checked for by PCR amplification with appropriate oligonucleotide primers.
Construction of nifL- and nifA-containing plasmids.
Oligonucleotides flanking the coding region of nifA and nifL were designed (see Table 2) to amplify the corresponding genes by PCR. For nifA, a 1634 bp promoterless DNA fragment was amplified and cloned as a HindIII fragment into the Km resistance gene of pVK100 under the control of the km promoter for pVA3 and in the opposite orientation for pVA1. For nifL, the amplified 1899 bp fragment contained 239 bp of the non-coding sequence; it was cloned as a HindIII–XhoI fragment into the Km resistance gene of pVK100 in the same orientation as the Km resistance gene, to yield pVL.
Yeast two-hybrid analysis.
The oligonucleotides designed to amplify the fragments encoding the desired domains of NifA and NifL are listed in Table 2. PCR amplification of strain A1501 genomic DNA was performed with the Proof Start DNA polymerase (Qiagen) and the resulting amplicons were cloned in-frame into pGBD-C1 or pGAD-C1 vectors (Table 1). The fusion junctions were verified in all the constructed plasmids by nucleotide sequence analyses. S. cerevisiae competent cells were prepared using the method described in the Matchmaker II protocol and pGAD and pGBD derivatives were co-transformed into the yeast recipient. The interaction between NifA and NifL domains was screened for by growth on SD medium lacking Leu, His, Trp and Ade (Table 3). The filter and quantitative liquid 
-galactosidase assay were done according to the protocol described by the Matchmaker II system (Clontech). Activity is expressed as Miller units (Miller, 1972).
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) and the amplicon was cloned into pET28a(+) vector (Novagen) to generate a His6-tagged NifA fusion protein. Similarly, oligonucleotides pnifL-C, F and R were used to amplify the C-terminal portion of nifL, which was constructed to fuse in-frame with GST (glutathione S-transferase) in the pGEX-2T vector (Pharmacia). Cultures containing the recombinant plasmids were grown aerobically in LB broth and expression from the T7 promoter was induced by addition of 1 mM IPTG. His6-NifA fusion protein was purified by nickel affinity chromatography on Hi-trap chelating columns (Pharmacia) and GST-NifLc fusion protein was purified by batch elution from affinity glutathione-Sepharose 4B beads (Pharmacia) as recommended by the manufacturer. SDS-PAGE of protein samples was performed by standard methods (Sambrook & Russell, 2001).
Analysis of protein complexes.
The pull-down assay was performed according to the method of Hasan et al. (2004). Purified GST-NifLc (10 ng) was immobilized on glutathione-Sepharose 4B beads and washed thoroughly with wash buffer (20 mM Tris/HCl pH 7.5, 2 mM EDTA, 100 mM NaCl, 12 mM -mercaptoethanol and 0.1 % Triton X-100). Immobilized GST-NifLc was then incubated with the purified His6-NifA for 1 h at 4 °C. After washing, bound proteins were eluted in SDS-PAGE sample buffer and separated by 8 % SDS-PAGE. Western blotting with anti-histidine or anti-GST antibodies (Invitrogen) was performed by standard methods (Sambrook & Russell, 2001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To further investigate the role of NifL in the regulation of nif gene expression in P. stutzeri, we used strain 1507B, in which most of the nifL coding sequence (1506 bp ClaI fragment) was substituted by a KIXX cassette cloned in the same orientation as the nifA gene. The non-polar effect of the nifL mutation on nifA expression was first checked by RT-PCR. A nifA transcript was indeed detected, both in the wild-type and in the 1507B nifL mutant, while no nifA transcript was produced in the nifL polar mutant constructed for this work, 1507A (Fig. 1a). The lack of nifA transcript in the nifL mutant 1507A strongly suggested that, as in other nitrogen fixers containing a nifL gene, nifA and nifL were transcribed from the same promoter in strain A1501. In addition, in strain 1507B the presence of nifA transcript was consistent with nifA transcription from the km promoter of the cassette.
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). It was then possible to investigate the effect of the increased production of NifA and NifL on nitrogenase activity by complementation analysis with plasmids expressing nifA or nifL. Expression of nifA in pVA3 was monitored from the km promoter of the vector. Indeed, pVA3 restored 30 % nitrogenase activity to the nifA-km mutant (1506) and nifL-km polar (1507A) strains (Fig. 1b), but the complementation reached up to 60 % when kanamycin was added to the culture medium (not shown). As expected, pVA1, in which nifA was cloned in the opposite orientation, did not affect nitrogen fixation (Fig. 1b). Plasmid pVA3 also increased nitrogenase activity of the wild-type and of the nifL non-polar mutant 1507B (Fig. 1b). In contrast, plasmid pVL, which carries the nifL gene, decreased nitrogenase activity of the wild-type and of the nifL non-polar mutant (Fig. 1b). Thus, NifL negatively modulates NifA activity. This is consistent with a role for NifL as NifA antiactivator.
Physical interaction between NifL and NifA in vivo
Deduced translation products of A1501 NifA and Azotobacter NifA share 82 % identity, suggesting that the domain structure of the P. stutzeri protein is very similar to that defined in Azotobacter NifA, and hence in other NifAs (Studholme & Dixon, 2003). This enabled us to design appropriate oligonucletides that could amplify portions encoding the N-terminal (NifAn), central (NifAm) and C-terminal (NifAc) domains, respectively, as schematized in Fig. 2. The same was applied to NifL, which shared 72 % identity with Azotobacter NifL. In that case, the nifL gene was divided into only two regions, encompassing the N-terminal PAS (NifLn) and the C-terminal (NifLc) domains, respectively (Fig. 2). Each of the domains was cloned into the yeast pGAD vector, which carries the activating domain of the yeast GAL4 transcriptional activator and into the pGBD vector, which carries the DNA-binding domain of GAL4. After co-transformation into yeast strain PJ69-4A, an interaction between the peptides fused to the pGAD and pGBD vectors is required so that the transcription from the promoters under the control of GAL4 can proceed (Table 3). As one of the reporter genes is lacZ, an enzymic assay for -galactosidase gives an estimation of the relative strength of the interaction (James et al., 1996). Fig. 3 shows an example of the growth on selective media and Table 3 summarizes the data obtained for growth and -galactosidase activity. From these data it is concluded that an interaction is detected between the entire NifA and NifL, and that the binding is limited to the C-terminal part of NifL and the central domain of NifA.
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), and the GST moiety of 27 kDa. The migration of the His6-NifA product, estimated at 64.2 kDa, was similar to that of the GST-NifLc fusion (Fig. 4). However, the two fusion proteins could be easily differentiated using anti-histidine or anti-GST antibodies after Western blotting, as shown below.
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. Lanes 1 and 3 correspond to control experiments performed only with NifA (lane 1) or with NifLc (lane 3), while lane 2 contained the elution product after the complex formation between NifLc and NifA. Fig. 4(a) shows protein staining. Anti-GST antibody, shown in Fig. 4(b), revealed material containing the GST-NifLc in lane 3 but not in lane 1, whereas anti-His antibody, in panel (c), revealed material containing His6-NifA in lane 1 but not in lane 3. Both antibodies revealed a protein product in lane 2 (Fig. 4b, c), suggesting that it corresponded to a mixture of NifA and NifLc. This is strongly in favour of complex formation between NifL and NifA, suggesting a specific binding between NifA and NifLc. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In this report, assay for nitrogenase activity of the nifL non-polar mutant 1507B showed it displayed a significant residual activity, suggesting that NifL is not an essential product for nitrogen fixation in strain A1501. This result is similar to that reported first in the case of K. pneumoniae (see Filser et al., 1983) and subsequently found in other strains containing a nifL gene, such as Azotobacter vinelandii (Blanco et al., 1993) and Azoarcus sp. (Egener et al., 2002). In addition, the decrease of nitrogenase activity observed both in the wild-type and in the nifL non-polar mutant in the presence of NifL excess produced by plasmid pVL (Fig. 1) is consistent with previous observations in K. pneumoniae (Buchanan-Wollaston et al., 1981). Thus, NifL most likely acts in modulating NifA activity in A1501, and this suggests that NifL is an antiactivator of NifA activity.
The yeast two-hybrid system is a useful technique to detect protein–protein interaction (James et al., 1996). It has been successfully used to explore binding between several components of the ntr and nif regulatory systems (Lei et al., 1999; Martínez-Argudo et al., 2002; Rudnick et al., 2002; Pawlowski et al., 2003; Chen et al., 2005). Thus, a direct protein–protein binding was detected between NifA and NifL in K. pneumoniae, Azotobacter vinelandii and Enterobacter cloacae (Lei et al., 1999; Martínez-Argudo et al., 2002; Liao et al., 2002). Results reported in P. stutzeri reveal also a direct protein–protein interaction between NifA and NifL and more precisely between the NifL GHKL (NifLc) and NifA AAA+ (NifAm) domains. Indeed, NifL PAS domain (NifLn) does not display binding activity to NifA (Table 3).
A prerequisite to further study of the mechanisms of interaction between NifL and NifA is to obtain the protein products in a soluble form (Lee et al., 1993). In this work we used convenient expression vectors to overproduce His-tagged NifA and GST fusion to NifLc. This enabled a rapid purification of both proteins and allowed us to demonstrate in vitro complex formation between NifLc and NifA (Fig. 4). This is consistent with the in vivo binding observed. It also suggests that under conditions not compatible with nitrogen fixation, inactivation of NifA is probably due to a protein complex between NifL and NifA so that nif genes cannot be transcribed and hence nitrogenase cannot be synthesized.
Mechanisms by which NifL and NifA modulate nif gene expression have been mainly studied in K. pneumoniae and A. vinelandii. The activity of NifLA complexes is modulated by GlnK (a PII parologue protein), ATP/ADP ratio and 2-oxoglutarate, but it is clear that mechanisms of signal communication between NifA and NifL are different in these two species (Martínez-Argudo et al., 2004). In both systems, GlnK, which senses the nitrogen status of the cell, was shown to interact with the NifLA via direct protein–protein interaction (Stips et al., 2004; Martínez-Argudo et al., 2004). In the case of Azotobacter, when nitrogen is limiting, GlnK, in its uridylylated form, does not interact with NifL, and thus NifL does not antagonize NifA activity (Martínez-Argudo et al., 2004). In contrast, GlnK is required for the relief of NifL inhibition in K. pneumoniae (He et al., 1998; Stips et al., 2004). In the present report, we have limited our investigation to the demonstration of an interaction between NifL and NifA, using both in vivo and in vitro techniques. It is not known whether a PII protein is involved in the modulation of NifLA activity. P. stutzeri A1501 carries a single copy of a glnB-like gene (this laboratory, unpublished observation), as in Azotobacter. The inactivation of the glnB-like gene and the purification of its protein product is in progress. Although P. stutzeri nifL is not an essential gene for nitrogen fixation, the NifL protein plays a regulatory role as antiactivator of NifA activity. The purification of both NifA and NifL proteins opens interesting perspectives to further study the mechanisms of interaction of NifL and NifA in this species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 May 2006;
revised 30 August 2006;
accepted 1 September 2006.
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