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Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand
Correspondence
Clive W. Ronson
Clive.Ronson{at}otago.ac.nz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: BIOMERIT Research Centre, Microbiology Department, University College Cork, Ireland.
Present address: Research and Enterprise Office, Centre for Innovation, University of Otago, PO Box 56, Dunedin, New Zealand.
The GenBank/EMBL/DDBJ accession numbers for the Asp718 fragments containing the nif/fix genes of Rlt NZP514 and ICC105 are EF165535 and EF165526, respectively. Accession numbers of the nifH–fixA intergenic regions of Rlt 144, 162C11, ABH12-1, ARH2-1, ARR13-1, ART1-2, AZR13-1, ICMP4074b, ICC144, NZP560, NZP561 and OSS82 are EF165519, EF165520, EF165521, EF165522, EF165523, EF165524, EF165525, EF165530, EF165531, EF165532, EF165533 and EF165534, respectively.
Two supplementary figures showing electron micrographs of nodules formed on WC by the ICC105 and NZP561 strains and nucleotide sequence alignment of the nifH–fixA intergenic regions of several CC and WC rhizobia strains are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Some rhizobia have a broad host range, such as Rhizobium sp. strain NGR234, which nodulates legume species from 122 genera, and the non-legume Parasponia (Pueppke & Broughton, 1999), while others have a narrow host range, such as Rhizobium leguminosarum biovar trifolii (Rlt), which only forms nodules on Trifolium species (Dénarié et al., 1992). In most instances, interactions are not established between non-compatible legume hosts and microsymbionts, because they fail to recognize each other. In some cases, however, the interaction proceeds as far as nodule development, but the microsymbionts do not enter the nodules or are not released from infection threads, or intracellular release does occur but the bacteroids fail to fix nitrogen. Host-dependent ineffective nodulation is particularly evident amongst Rlt strains that nodulate clover species. For example, some Rlt strains form effective (nitrogen-fixing) nodules on Trifolium repens, Trifolium pratense and Trifolium subterraneum, whereas other strains are effective on T. repens and T. pratense but ineffective on T. subterraneum, and a third group of strains are effective on T. subterraneum but only partially effective or ineffective on T. repens and T. pratense (Greenwood, 1964). The genetic bases of the host-specific nitrogen fixation phenotypes are not understood.
Trifolium ambiguum (Caucasian clover, CC) is a forage legume with a number of beneficial characteristics, including disease resistance and rhizomatous habit, which enable growth under harsh environmental conditions (Bryant, 1974; Speer & Allinson, 1985). It is endemic throughout the Caucasus and Eastern Europe, and is well adapted to the cold, dry conditions of the high mountainous slopes and steppes of these regions (Taylor & Smith, 1998). It has very specific rhizobial requirements and strains capable of forming effective nodules are not found outside its region of origin. There are currently very few symbiotically effective strains that have been isolated and evaluated for use as commercial inoculants (Beauregard et al., 2003, 2004; Hely, 1963; Pryor et al., 1998; Zorin et al., 1976; Zorin & Hely, 1976).
CC is being introduced into New Zealand pastures in areas where growth of T. repens (white clover, WC) may be limited by environmental conditions that CC is better adapted to (Watson et al., 1996). Rlt strain ICC105 and other Rlt strains able to form effective (Fix+) nodules on CC also form nodules on WC; however, these nodules are ineffective (Fix–). This effect is of potential agronomic importance in New Zealand, where pastoral agriculture is largely based on WC/ryegrass pastures. Widespread use of CC will likely lead to the build-up of significant populations of CC rhizobia in soils with the potential to adversely affect WC growth (Elliot et al., 1998; Pryor et al., 2004).
The aims of this study were to decipher the genetic basis of the ineffective nodulation response of ICC105 on WC, and to construct an Rlt strain effective on both clovers that might be suitable for development as an inoculant strain. The expression of the nif and fix structural genes, required to form a functional nitrogenase enzyme, was examined.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Strain NZP561 is a commercially used WC inoculant strain, while ICC105 is used as a CC inoculant strain. The plasmid pPN1 is a cointegrate of the WC Rlt strain NZP514 symbiotic plasmid pRtr-514a and P-group plasmid R68.45 (Scott & Ronson, 1982). Both NZP561 and NZP514 are fully effective on WC. However, the symbiotic properties of strain NZP514 are unstable due to the instability of pRtr-514a (Scott & Ronson, 1982), and the Sym+ NZP514 strain was not available to us. Hence, strain NZP561 was used as the control strain for plant tests. R. leguminosarum strains were grown at 28 °C in tryptone yeast extract (TY) or rhizobium defined medium with 10 mM glucose (G/RDM) (Hubber et al., 2004). Escherichia coli strains were grown in LB or TY medium. Antibiotics were added at the following concentrations: for E. coli, 15 µg tetracycline ml–1, 50 µg kanamycin ml–1, 100 µg ampicillin ml–1 and 25 µg gentamicin ml–1; and for R. leguminosarum, 2 µg tetracycline ml–1, 50 µg neomycin ml–1 and 50 µg gentamicin ml–1. Plasmids were transferred from E. coli strains to R. leguminosarum strains using biparental or triparental matings, as described previously (Hubber et al., 2004).
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). Visualization of large plasmids was carried out by Eckhardt gel analysis, as described elsewhere (Hynes & McGregor, 1990). For DNA sequencing, restriction fragments were cloned into pUC19 and sequenced using the M13 forward and reverse primers and custom primers. The sequence was assembled using Vector NTI Advance 10 (Invitrogen). ORFs were identified using GeneMark.hmm with a heuristically derived model (Besemer & Borodovsky, 1999) and through database searches using BLAST (Altschul et al., 1997). Nucleotide and protein sequences were aligned using the CLUSTAL W option within MEGALIGN (DNASTAR). Rhizobium genomic DNA was extracted as described previously (Sullivan et al., 1995). PCR was carried out using the Phusion High-Fidelity PCR kit (Finnzymes) as recommended by the manufacturer. The plasmid vector pIJPAR was constructed by cloning a KpnI/BamHI fragment containing the parDE plasmid stability locus from pRT101 into pIJ3200. To construct the plasmid vector pFJX, a DNA fragment containing the lacZ gene and cloning cassette was released from pFUS2 by digestion with StyI and the ends blunted with Klenow enzyme. The fragment was then digested with HindIII and cloned into pFAJ1700 digested with HindIII and EcoRV.
Construction of pPN1 cosmid library.
Plasmid DNA was prepared from E. coli HB101 carrying pPN1 using a Plasmid Maxi kit (Qiagen), partially digested with Sau3A and size fractionated on an 0.4 % agarose gel. DNA fragments between 25 and 30 kb were recovered from the gel using a QiaexII gel extraction kit (Qiagen) and ligated into BamHI-digested pIJ3200. The ligation mixture was packaged into 
phage heads using the Packagene Lambda packaging system (Promega), and the packaged cosmids were used to infect E. coli HB101. Transfectants were selected on LB medium containing tetracycline.
Mutagenesis.
E. coli HB101 strains carrying cosmids pSHM58 or pSHM74 were mutagenized with Tn5, as previously described (Sullivan et al., 2001). For the creation of an ICC105 nifA mutant, a PCR product containing the ICC105 nifA gene was amplified using primers incorporating PstI sites (FixX and NifB, Table 2), digested with PstI, and ligated into pIJ3200 to create pSHM101. The pSHM101 plasmid was mutagenized using an in vitro transposon EZTn-GN, and the positions of insertion were mapped by sequencing, as previously described (Hubber et al., 2007). The plasmid pSHM101nifA : : EZTn-GN, which carried an EZTn-GN insertion at nucleotide 453 of nifA, was mated into ICC105, and marker exchange of the transposon insertion was forced by plasmid incompatibility using pPH1JI, as described previously (Hubber et al., 2007). The strain was cured of pPH1JI by introduction of pLAFR1 and selection of GmS TcR colonies, generating strain SHM103. Replacement of the ICC105 nifH/fixA interpromoter region with the 
Kan interposon was achieved using PCR products amplified using the primer pairs Nif KanL/Nif KanR and Fix KanL/Fix KanR (Table 2), and the Kan cassette was excised from pHP4 Kan. The resulting DNA replacement cassette was subcloned into pJQ200SK that had been digested with SpeI and XbaI, creating pSHM119. The pSHM119 plasmid was transferred to ICC105 and marker exchange forced, as described previously (Hubber et al., 2004), creating strain SHM119. Strain SHM126 was created by the same method, except that the Kan cassette was omitted. Deletion of the nifH/fixA interpromoter region in sucrose-resistant colonies was confirmed by PCR using the primers NifHXP and FixAXP.
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Recombination of the Rlt NZP514 nif/fix genes into ICC105.
An 18 kb fragment was released from pSHM74 by digestion with Asp718, blunted with Klenow enzyme and cloned into the SmaI site of pJQ200SK, creating pSHM122. This plasmid was transferred into ICC105, and integration of pSHM122 in the transconjugants was confirmed by Southern hybridization analysis. Two of the resulting GmR single-crossover recombinant strains (SHM122a and SHM122f) which formed Fix+ nodules on both WC and CC were plated on RDM agar containing 5 % sucrose to detect strains that had undergone a second homologous recombination event that excised the vector. GmS strains designated SHM122a1–SHM122a16 and SHM122f1–SHM122f16 were selected for further study.
Transmission electron microscopy.
Nodules were excised from plants 21 days after inoculation and fixed, sectioned and stained as previously described (Hubber et al., 2004). They were examined with an Akashi 002A microscope at 100 kV accelerating voltage.
Plant assays.
Plant studies were performed using WC (T. repens cv. Grasslands Huia) and CC (T. ambiguum cv. Endura), as described elsewhere (Vincent, 1970). Seeds were surface-sterilized and germinated on inverted 0.8 % water agar plates. Germinated seedlings were transferred to Jensen's agar slopes in glass test tubes (18 mm diameter for WC and 25 mm diameter for CC). For each strain, 10 plants were inoculated by streaking the bacteria below the tip of the lengthening radicle. Plants were cultivated under controlled environmental conditions of 70 % humidity; 22–25 °C, 16 h (day); 14 °C, 8 h (night). Plants were harvested at 4 weeks post-inoculation, and the effectiveness of the symbiosis was determined by measuring the wet weight of foliage above the first cotyledonary node (Vincent, 1970).
Rhizobia were recovered from nodules at 4–6 weeks post-inoculation, as previously described (Sullivan et al., 1995). The nodule suspension was plated for single colonies on G/RDM, and colonies were then patched onto G/RDM media containing appropriate antibiotics to test for cosmid retention. β-Galactosidase assays were performed as described by Miller (1972), using bacteroid suspensions prepared from 14-day-old nodules harvested from 10 plants per strain.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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To delineate the region of pPN1 required by ICC105 for effective nodulation of WC, a cosmid library prepared from pPN1 DNA was transferred en masse into ICC105 and the resulting transconjugants screened on WC. Six transconjugants, including strain SHM58, formed Fix+/– nodules (12.5–81 % mean wet shoot weight compared to a Fix+ NZP561 control). A mixture of large pink and small white nodules was observed on WC roots inoculated with the six transconjugants. The average percentage of rhizobial colonies isolated from the nodules that were TcR was 68 % for pink nodules and 12.5 % for white nodules. This suggested a correlation between cosmid stability and effectiveness on WC. All of the transconjugants remained Fix+ on CC.
Comparison of EcoRI, HindIII, PstI and SalI restriction profiles of cosmids isolated from the six Fix+/– transconjugants identified several conserved fragments. An 18 kb Asp718 fragment that spanned this conserved region and a 12 kb Asp718/BspLU11I subfragment of this region were subcloned from one cosmid, pSHM58, into the stable broad-host-range vector pIJPAR to create pSHM74 and pSHM75, respectively (Fig. 1). These plasmids were introduced into ICC105, creating strains SHM74 and SHM75, which were screened on WC and CC. Both strains produced Fix+ nodules on CC, while on WC, SHM74 produced Fix+ nodules and SHM75 produced Fix+/– nodules (61.7 % mean wet shoot weight compared to Fix+ NZP561 control).
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). Analysis of the sequence indicated that nifH, nifD, nifK, fixA, fixB, fixC, fixX, nifA nifB, fdxN and fixU genes were sufficient to enable ICC105 to form Fix+/– nodules on WC, and that at least some of the remaining genes that were present on pSHM74 but absent from pSHM75 (Fig. 1) were required for a fully Fix+ phenotype.
Tn5 mutagenesis of pSHM58
To identify genes required to convert ICC105 to Fix+ on WC, pSHM58 was mutagenized using Tn5, and the resulting population of plasmids was screened to find plasmids that no longer converted ICC105 to a Fix+ phenotype on WC. All insertions in the nifH, nifD, nifK, fixA, fixB, fixC and nifB genes produced transconjugant ICC105 strains with a Fix– phenotype on WC, whereas insertions in nifE and nifN produced a Fix+ or Fix+/– phenotype (Fig. 1). All insertions in the nifA gene were Fix+, as were several insertions mapped by restriction analysis to the region downstream of nifN (data not shown). Tn5 insertions were not isolated in trxA, fixX, fdxN or fixU.
The ICC105 nifA gene is functional in WC nodules and its expression is not limiting
The above results suggested that ICC105 required the structural nif/fix genes of NZP514 but not the regulatory nifA gene for nitrogen fixation on WC. This suggested that the ICC105 nifA gene was expressed and its product was functional within WC nodules, but that the ICC105 structural nif/fix genes were not. To further investigate this surprising finding, an ICC105 nifA mutant strain SHM103 was constructed. As expected, the strain was Fix– on both WC and CC. pSHM58 and its derivatives containing Tn5 insertions within the NZP514 nifA, fixA or nifH genes were then mated into strain SHM103 to give strains SHM107, SHM104, SHM105 and SHM106, respectively. The nitrogen-fixation phenotypes of these strains were determined on WC and CC (Table 3). Strain SHM107 was Fix+ on both clovers, indicating that when both the ICC105 and NZP514 nif and fix genes were present, the NZP514 NifA was able to activate either set on both CC and WC. As expected, SHM104, which lacks a functional nifA gene, was Fix– on both clovers. Strains SHM105 and SHM106 were Fix– on WC, indicating either that the NZP514 NifA was incapable of activating transcription of the ICC105 nif and fix genes in WC nodules or that the ICC105 nifH and fixA genes were unable to complement NZP514 nifH and fixA mutants. SHM105 and SHM106 were partially effective on CC (25.1 and 34.3 % mean wet shoot weight, respectively, compared to a Fix+ ICC105 control), indicating that the NZP514 NifA was at least partially functional in CC nodules.
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), and strongly suggest that the quantity of NifA is not limiting for nif/fix gene expression.
Comparison of the ICC105 and NZP514 nif and fix genes
Further sequence analysis was carried out in order to identify any features which might indicate why the ICC105 nif and fix genes were non-functional in WC nodules. A 17.7 kb Asp718 fragment of ICC105 containing nif and fix genes was identified using Southern hybridization analysis with probes derived from pSHM74. This fragment was cloned and sequenced (Fig. 1b). The analysis showed that ICC105 contained nifH, nifD, nifK, nifE, nifN, fixA, fixB, fixC, fixX, nifA, nifB, fdxN and fixU genes organized in the same way as those found in NZP514. Alignments of the ICC105 and NZP514 Nif and Fix proteins showed percentage identities ranging from 82.2 % for NifB to 97.3 % for NifH. The NifA proteins from the two strains were strongly conserved and, like the NifA of Rlt strain ANU843 (Iismaa & Watson, 1989), both lacked the N-terminal region found in NifA of other rhizobia. When the 5' regions of nifA from ICC105, NZP514 and ANU843 were aligned, it was found that ICC105 had the nucleotides GGG at the ATG start codon position assigned for ANU843; however, all three strains had a GTG codon located 21 bp upstream of the ATG/GGG site, and the intervening sequences, encoding seven amino acids, were identical in all three strains. In each case, the GTG codon was preceded by a potential ribosome-binding site (GGAG), and hence it may be the translational start site of nifA in all three strains.
The putative promoter regions located upstream of nifH, fixA, nifA and nifB in ICC105 and NZP514 were also highly conserved. One notable difference was that the RpoN-binding site that precedes nifH in NZP514 contained a GA at the –12 position as opposed to the GC consensus (Fig. 2). The most obvious difference between the nif/fix regions of ICC105 and NZP514 was between the divergent promoters of the nifH and fixA genes. The ICC105 nifH/fixA interpromoter region contained a 111 bp region that was not present in NZP514 (Fig. 2). Although no functional motifs or promoter elements, such as NifA-binding sites, were identified within this region, its proximity to the nifH and fixA promoters suggested that it has some regulatory role.
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) and sequenced to examine their similarity. The strains examined (Table 1) were either Fix+ on WC (Rlt NZP560, OSS82, ABH12-1, AZR13-1) or Fix+ on CC (Rlt ICC105, 144, 162C11, ICMP4074b, ARR13-1, ART1-2, ARH2-1). Alignment of the sequences (Supplementary Fig. S2) demonstrated that all strains that were Fix+ on CC contained the extra 111 bp region, and four also contained an extra 18 or 19 bp within this region, while all strains that were Fix+ on WC lacked the region. All WC strains had the GA doublet at the –12 position of the nifH promoter, whereas all strains effective on CC had GC.
Deletion of the extra 111 bp sequence in the ICC105 nifH/fixA interpromoter region
The above results suggested that the extra 111 bp nifH/fixA interpromoter region in ICC105 played a role in the host-specific nitrogen-fixation phenotype. To test this hypothesis, two mutants were created in which the region (see Fig. 2) was either replaced by the Kan interposon (strain SHM119) or deleted without replacement by a marker (strain SHM126). Both strains had an identical nitrogen-fixation phenotype to that of wild-type ICC105 on both WC and CC. Therefore, the extra 111 bp nifH/fixA interpromoter region apparently did not act negatively upon the expression of the nif/fix genes in WC or positively in CC.
Exchange of the ICC105 nifH–fixA intergenic region with that of NZP514
To further investigate the possibility that a part of the nifH/fixA intergenic region was a determinant of the host-specific nitrogen-fixation phenotype, ICC105 derivatives were constructed with the nifH/fixA intergenic region from ICC105 replaced with that from NZP514. To do this, the 18 kb Asp718 fragment of pSHM74 (Fig. 1a) was subcloned into pJQ200SK to give pSHM122, which was recombined by single crossover into the ICC105 genome in order to create hybrid strains carrying both ICC105 and NZP514 nif and fix DNA. The symbiotic phenotype of six hybrid strains was determined on both WC and CC. One strain was Fix– on WC and Fix+/– on CC, three were Fix+ on WC and Fix± on CC, and two were Fix+ on both clovers. A second recombination event that excised the vector was then detected by sucrose selection in the two strains that were Fix+ on both clovers (SHM122a and SHM122f), and the symbiotic phenotypes on CC and WC of 32 of the resulting strains were determined. Of these, 29 had a phenotype identical to that of ICC105, i.e. Fix+ on CC and Fix– on WC. Southern hybridization analysis of genomic DNA isolated from two of these strains, SHM122f4 and SHM122f5, using the 18 kb Asp718 fragment purified from pSHM74 as a probe, demonstrated that only part of the NZP514 nifKE genes had recombined in each case (Fig. 1b). The remaining three recombinant strains, SHM122a5, SHM122a11 and SHM122f7, were fully Fix+ on WC and Fix– on CC, i.e. the opposite phenotype to that of ICC105. Southern hybridization analysis of these strains demonstrated that a much larger region had recombined, encompassing in all three strains the nifH–fixA intergenic region, and at least the nifHD and fixAB genes and part of the nifK and fixC genes (Fig. 1b). This result further supported the hypothesis that the differences between ICC105 and NZP514 within the nifH–fixA intergenic region determine the host-specific expression of the nifHDKEN and fixABCX operons on either side.
The ICC105 nifH gene is not expressed in WC nodules
Strain SHM109d was used to determine whether the host-specific phenotype was due to lack of ICC105 nif/fix gene expression as opposed to lack of protein function in WC nodules. This strain has a pFUS2 construct integrated such that the lacZ gene was fused to the native nifH promoter and the cloned nifH promoter region was fused to the native nifH coding sequence. The strain formed Fix+ nodules on CC and Fix– nodules on WC. The nifH–lacZ fusion gene was expressed in CC nodules (175.6±6.7 Miller units compared to 6.4±0.6 Miller units for ICC105 control) but not in WC nodules (0.6±0.1 Miller units compared to 3.1±0.2 Miller units for ICC105 control).
Effect of DNA upstream of the promoter on ICC105 and NZP561 nifH expression
To determine whether the DNA present upstream of the nifH promoter in the CC rhizobia but not in WC rhizobia affected nifH expression, strains SHM127 to SHM137 were used. Strains SHM127 to SHM132 are ICC105 derivatives containing pFJX-based plasmids with the ICC105 nifH 5' region plus upstream DNA of varying length fused to lacZ, while strains SHM133 to SHM137 are similar NZP561 derivatives but with NZP561-derived nifH–lacZ fusions (Fig. 3). DNA from strain NZP561 rather than NZP514 was used to create the latter fusions to ensure appropriate homologous recombination, although the fixA–nifH intergenic regions in the two strains are very similar (see Supplementary Figure S2). The expression of lacZ was assayed in bacteroids isolated from 2-week-old nodules of WC (all strains) and CC (SHM127–SHM132) (Fig. 3). Expression from the NZP561 nifH promoter in Fix+ WC nodules was high (748.2–935.2 Miller units above negative control) and unaffected by the inclusion of further DNA upstream of the NifA-binding site (Fig. 3, strains SHM134–136). Similarly, consistent expression levels (314.2–405.6 Miller units above negative control) were observed from the ICC105 nifH promoter in Fix+ CC nodules, regardless of the length of the region upstream of the NifA-binding site (Fig. 3; strains SHM128–SHM131). In contrast, expression from the ICC105 nifH promoter in Fix– WC nodules was dependent on the extent of the region upstream of the NifA-binding site. Inclusion of the region between the NifA-binding site and the extra 111 bp region caused a 41 % reduction in nifH expression (SHM129). Addition of the 111 bp region did not cause a further reduction in expression (SHM130), but inclusion of the entire nifH–fixA intergenic region caused a 73 % reduction in nifH expression (SHM131). Strains showed wild-type phenotypes with respect to nitrogen fixation on WC and CC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The genetic organization and sequences of the NZP514 and ICC105 nif and fix gene regions were very similar to each other and to that of Rlt strain ANU843 (Iismaa et al., 1989), with the notable exception of a 111 bp insertion in the nifH/fixA intergenic region of ICC105 compared to the WC strains. Several lines of evidence suggest that the differences in the nifH–fixA intergenic region are involved in the host-specific control of gene expression. The nifH and fixA operons are expressed from divergent promoters in this region and neither was active in WC nodules formed by ICC105. The 111 bp insertion was present in all rhizobia tested that were able to form Fix+ nodules on CC. Conversely, it was absent from all strains tested able to form Fix+ nodules on WC. Most importantly, replacement of the ICC105 nifH–fixA intergenic region and a portion of the flanking nif/fix genes with analogous genes from WC strain NZP514 produced strains that were Fix+ on WC and Fix– on CC, i.e. had a reversed phenotype. Finally, as discussed below, expression studies indicated that the full intergenic region acted to inhibit ICC105 nifH expression in WC nodules.
The regulation of nifH expression has been extensively studied in rhizobia and in all cases is under the control of the NifA protein that interacts with the RpoN sigma factor to activate gene expression. Regulation is at the level of nifA expression and NifA activity and, in some cases, the expression of a symbiosis-specific copy of the rpoN gene (Dixon & Kahn, 2004; Fischer, 1994). No other proteins directly involved in nifH regulation have been described. One potentially important difference in the nifH/fixA intergenic region was that all WC strains, including strains ANU843 (Iismaa et al., 1989) and SU329 (Scott et al., 1983), had a non-canonical GA doublet at the conserved –12 position of the RpoN-binding site of the nifH promoter, whereas all strains effective on CC had the canonical GC doublet. The nifH promoters of Rhizobium etli (Valderrama et al., 1996) and Mesorhizobium loti (Sullivan et al., 2002) also have the GA doublet, and in the case of R. etli, it has been shown that nifH expression requires the symbiosis-specific RpoN protein (Michiels et al., 1998). However, since both WC and CC strains had GC at the –12 position of the fixA promoter, and ICC105 required the WC strain fixA operon to fix nitrogen on WC, it seems unlikely that the lack of GA at the –12 of the ICC105 nifH promoter is involved in the host-specific phenotype on WC.
The hypothesis that the additional 111 bp region present in the nifH–fixA intergenic region in CC strains was solely responsible for the lack of ICC105 nif/fix expression in WC nodules was eliminated by the finding that deletion of the region did not affect the phenotype of ICC105, i.e. the strain remained Fix– on WC and Fix+ on CC. Furthermore, the lacZ reporter gene studies of the nifH promoter implicated regions beyond the NifA-binding site and either side of the 111 bp extra region as important. A nifH–lacZ reporter gene present in cis to the wild-type ICC105 nifH gene (strain SHM109d) was not expressed in WC nodules, confirming that the block in nifH expression was at the transcriptional level. However, an in trans lacZ reporter fused to the nifH promoter consisting of sequence up to and including the NifA-binding site but with no additional DNA was expressed in WC nodules at 56 % of the level in CC nodules. The level in CC nodules was about double that of the in cis fusion, probably reflecting the additional copy number of the in trans fusion. Inclusion of the 127 bp region between the NifA-binding site and the extra 111 bp region caused a 41 % reduction in nifH expression in WC nodules (SHM129); however, addition of the 111 bp region did not cause a further reduction in expression (SHM130). Inclusion of the entire nifH–fixA intergenic region caused a 73 % reduction in nifH expression (SHM131), consistent with the lack of expression of the in cis fusion. Hence, the further reduction in expression in SHM131 compared to SHM129 is unlikely to be due to the quantity of NifA or RpoN becoming limiting as a consequence of sequestration at the binding sites present on the fixA promoter. Furthermore, a similar reduction in expression was not observed in CC nodules. It seems most likely that the reduction was due to additional host-specific regulatory elements located between the 111 bp extra region and the fixA translational start site, a hypothesis that fits with the observation that ICC105 fixA expression was also under host-specific regulation.
There are reports in the literature that suggest that regulatory mechanisms in addition to NifA/RpoN may act on nif gene expression in rhizobia. Timmers et al. (2000) found that when NifA was constitutively expressed in a Sinorhizobium. meliloti strain within alfalfa nodules at oxygen concentrations conducive to NifA activity, this did not lead to constitutive expression of the nifH gene. Furthermore, nifH was not expressed in certain zones of nodules formed by wild-type bacteria, despite strong nifA expression. They propose that nifH expression is not only subject to regulation by oxygen concentration, via NifA, but is also under the control of an additional negative regulator (Timmers et al., 2000). Cebolla et al. (1994) found that expression of the S. meliloti nifH and fixA promoters under symbiotic conditions in heterologous rhizobial backgrounds was very low compared to expression in a homologous background (especially for fixA), suggestive of host-specificity. This effect of decreased expression was most strongly seen in an Rlt background. Wang et al. (1991) demonstrated that the S. meliloti nifH promoter contains a ‘downstream element’ located between the transcriptional start site and translational start codon that is specifically involved in nifH expression in microoxic conditions, thus demonstrating a potential regulatory site involved in differences between microoxic and symbiotic regulation of nifH in S. meliloti. In an experiment in which symbiotic plasmids were exchanged between Rlt and Rl biovar viciae, the ability to fix nitrogen on WC or pea was associated with the presence of the nif genes from the plant's normal rhizobial partner. A Rlt strain carrying both the Rlt and Rl bv. viciae nod genes but nif genes from only Rlt produced Fix+ nodules on WC but Fix– nodules on Pisum sativum, whereas a strain containing both sets of nif genes was effective on both hosts (Christensen & Schubert, 1983). The authors propose that ‘the biochemical signal necessary for activating the expression of the R. leguminosarum nif genes in pea nodules may be different from that required for expression of the R. trifolii nif genes in clover nodules’ (Christensen & Schubert, 1983). Taken together, these studies provide strong support for our hypothesis of an extra level of nifH/fixA gene regulation in response to the host plant.
The mechanism underlying host-specific nif gene regulation in rhizobia remains an area of speculation. Our data support the hypothesis that a signal from the plant is transmitted to a regulatory protein that either blocks or allows NifA activation of the nifH or fixA promoters, depending on the particular host. Such an effect cannot involve direct protein–protein interaction with NifA in the absence of DNA binding, as the ICC105 nifH promoter is active in WC nodules when separated from upstream DNA. We propose that a protein acting either in response to a host-specific signal or in the absence of such a signal is able to bind upstream of NifA-binding sites and interact with NifA to prevent it activating nif/fix gene expression. We are currently testing this hypothesis.
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ACKNOWLEDGEMENTS |
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Edited by: M. F. Hynes
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 12 February 2007;
revised 26 April 2007;
accepted 17 May 2007.
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