1 Departamento de Biotecnología, E. T. S. de Ingenieros Agrónomos, Universidad Politécnica de Madrid, 28040 Madrid, Spain
2 Consejo Superior de Investigaciones Científicas (C.S.I.C.), 28040 Madrid, Spain
Correspondence
Tomás Ruiz-Argüeso
ruizargueso{at}bit.etsia.upm.es
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ABSTRACT |
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INTRODUCTION |
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In nitrogen-fixing, endosymbiotic bacteria such as Bradyrhizobium japonicum and Rhizobium leguminosarum, H2 is generated by nitrogenase itself, and a particular coupling occurs between hydrogen oxidation and nitrogen fixation inside the legume nodules. Although B. japonicum contains a H2-sensing and signal transduction system, mediated by the proteins HoxA, HupT and HupUV, which allows these bacteria to express hydrogenase in free-living conditions (Van Soom et al., 1993b, 1999
; Black et al., 1994
), the induction of the hydrogenase system in soybean nodules does not require H2, and it has been proposed that the FixK2 protein could be the activator that co-ordinates hydrogenase and nitrogenase expression (Durmowicz & Maier, 1998
).
The expression of hydrogenase activity by R. leguminosarum bv. viciae has only been observed in symbiosis with legumes, and the regulation of this expression has been studied in detail (Ruiz-Argüeso et al., 2001). R. leguminosarum bv. viciae strain UPM791 contains a large gene cluster of 18 genes, hupSLCDEFGHIJKhypABFCDEX, which are needed for hydrogenase synthesis (Fig. 1
a). Although several transcriptional units were initially defined by symbiotic complementation analysis (Leyva et al., 1990
; Hidalgo et al., 1992
), only two major promoters have been characterized within this gene cluster. First, a NifA-dependent -24/-12-type promoter (P1), responsible for symbiotic activation of at least the hydrogenase structural genes hupSL, was identified upstream of hupS (Hidalgo et al., 1992
; Brito et al., 1997
). This NifA-dependent promoter constrains the expression of hydrogenase activity to symbiotic cells, and results in a temporal and spatial co-expression of nitrogenase and hydrogenase structural genes in pea nodules (Brito et al., 1995
). Second, an Fnr-type promoter (P5), located upstream of hypB within the hypA gene, activates the hypBFCDEX operon in pea bacteroids and also in vegetative cells in response to microaerobic conditions (Hernando et al., 1995
). Two copies of an fnrN gene have been identified in R. leguminosarum bv. viciae, and their transcription is autoregulated (Colombo et al., 2000
). FnrN controls both hydrogenase and nitrogenase activities by regulating the expression of the hypBFCDEX and fixNOQPfixGHIS operons, respectively (Gutiérrez et al., 1997
).
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METHODS |
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Plasmid construction.
To generate transcriptional hupGH : : lacZ fusions, a 2·3 kb XhoIKpnI fragment from cosmid pAL618 was cloned in vector pMP220 in both orientations with respect to the lacZ gene, yielding plasmids pHL320 and pHL330. Plasmid pSPD1 was generated by cloning a 1·6 kb HindIII fragment from pHL320 in vector pSPV4. Plasmids pSPD2, pSPD3 and pSPD5 contain deletion fragments that were generated by PCR using SPD2 (5'-ATTTGCGCGGCGAGTGAAAAGAGA-3'), SPD3 (5'-AGATGTTGGCGATATTTTCCT-3') and SPD5 (5'-ATCTGCCCTGGTCCGCGCCCTG-3') as upper primers, SPDR (5'-GCAACCCAAAAACCAGCCTTCA-3') as lower primer and pHL320 DNA as template. The resulting PCR fragments of 654, 600 and 464 bp, respectively, were cloned in pCR2.1-TOPO generating TOPO-SPD plasmids. Then, the 414, 360 and 224 bp HindIII fragments from TOPO-SPD2, TOPO-SPD3 and TOPO-SPD5 were fused to the lacZ gene in vector pSPV4, resulting in plasmids pSPD2, pSPD3 and pSPD5, respectively.
To generate plasmid vector derivatives pEK26 and pSKA, a 745 bp HindIIIKpnI fragment from the hupGH genes and the entire pMJ220 plasmid as an EcoRI fragment, respectively, were cloned in pBluescript vector. pSKDG was the plasmid resulting from cloning a 1·6 kb HindIII fragment that spanned the hupD to hupG genes and contained the P3 promoter into the pBluescript SK+ vector.
Transcription mapping.
The location of the 5' end of hupG mRNA was determined by primer extension analysis as described by Hidalgo et al. (1992). For this assay, two synthetic oligonucleotides, HUPFG2 (5'-TCATCGACGACAGGTAGGTG-3') and HUPFG3 (5'-TCGCCGGTGAAGAACAC-3'), were used. These primers were complementary to the mRNA sequence corresponding to amino acids 1420 and 3945, respectively, of HupG. Total RNA for this analysis was isolated from aerobic cells of E. coli ET8000(pHL320, pMJ220) and ET8000(pSPD5, pMJ220) as described previously (Summers, 1970
). The synthetic primers were labelled with [
-32P]dATP, and the corresponding DNA extension products obtained by reverse transcriptase reaction from RNA were visualized by autoradiography.
Site-directed mutagenesis of the P3 promoter.
Site-directed mutagenesis of the RpoN-binding sequence was carried out on plasmid pSKDG using the Quick ChangeTM Site Directed Mutagenesis Kit (Stratagene) and following the manufacturer's protocol. Two synthetic, complementary oligonucleotides carrying the corresponding mutations (underlined) were used to generate the promoter mutation. First, the oligonucleotide 5'-CTACTTTCCTCAGTCACCCACGCCGTTTGCAG-3' and its complementary primer were used to mutagenize the -24 position and, second, oligonucleotide 5'-CACCCACGCCGTCTCGAGATCATCATTCGC-3' and its complementary primer were used to introduce the mutation in the -12 position. To confirm the presence of correct mutations in the resulting plasmid, pSKP3, the P3 promoter region was sequenced. The 1·6 kb HindIII fragment carrying the mutated promoter was cloned in pK18mobsacB (Schäfer et al., 1994) together with an additional 1·5 kb HindIIIEcoRI DNA fragment containing the rest of the hupG gene, and the hupHIJ genes. The resulting plasmid, pK18P3, was introduced into R. leguminosarum UPM791 by conjugation, and the P3 promoter was replaced by double crossover using the sacB system. Correct replacement of the promoter was confirmed by Southern blot experiments using a 654 bp EcoRI fragment as DNA probe for the P3 promoter and taking advantage of the XhoI restriction site introduced with the mutation at the -12 position.
In situ hybridization of nif and hup mRNAs in pea nodules.
Root pea (Pisum sativum L. cv. Rondo) nodules were harvested 16 days after inoculation with R. leguminosarum bv. viciae strain UPM791 or derivative mutant AL51 (hupS). Nodule sections (7 µm thick) were prepared as described by Yang et al. (1991)
. The hupGH antisense and sense probes were prepared from pEK26. The nifH RNA antisense probe was obtained as described by Yang et al. (1991)
. The hupGH and nifH RNA probes were obtained and labelled in vitro with [35S]UTP using the appropriate (T3 or T7) RNA polymerase phage system (Van de Wiel et al., 1990
). Nodule sections were hybridized with the RNA probes and developed after 3 or 4 weeks exposure as described by Van de Wiel et al. (1990)
. Micrographs were taken in bright field and bright field with epipolarization.
Plant tests and enzyme assays.
Pea (P. sativum L. cv. Frisson) plants were used as the host for R. leguminosarum bv. viciae strains. Conditions for plant inoculation and growth have been described previously (Leyva et al., 1987), and plant nutrient solution was supplemented with 20 µM NiCl2. Nitrogenase derepression in K. pneumoniae was carried out as indicated by Imperial et al. (1984)
.
-Galactosidase activities in Rhizobium and pea bacteroids were determined as described by Miller (1972)
. Hydrogenase activity in bacteroid suspensions was measured by an amperometric method with oxygen as the terminal electron acceptor (Ruiz-Argüeso et al., 1978
). The protein content of bacteroid suspensions was measured by the bicinchoninic acid method (Smith et al., 1985
) after alkaline digestion in 1 M NaOH at 90 °C for 10 min and with BSA as a standard.
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RESULTS |
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No canonical NifA UASs were present in the P3 promoter. However, since NifA-dependent expression of the hup structural promoter (P1) has been shown to depend on binding of NifA to non-canonical sequences (Brito et al., 1997), the presence of such putative activating sequences was investigated by deletion analysis of the DNA region upstream of hupG. This analysis was carried out in E. coli cells in the presence of K. pneumoniae NifA. Fig. 3
shows that NifA-dependent P3 activity did not require any DNA sequence located upstream of the putative IHF motif, thus suggesting that NifA is activating P3 from solution. Similar results were obtained for the native levels of P3 activity in R. leguminosarum pea bacteroids. When the same deletion plasmids were introduced in R. leguminosarum, no effect of these deletions on P3 activity was observed (Table 2
), except for the deletion in plasmid pSPD5, which includes the
54-binding sequence.
Finally, the possible involvement of the putative IHF-binding sequence in P3 activation by NifA was ruled out after observing that the NifA-dependent -galactosidase activity of the hupGH : : lacZ fusion in pHL320 was not affected in E. coli SE1000, an IHF mutant (Table 5
). As control of IHF-dependent activation, the reporter activity associated with pHL315 (hupSL : : lacZ) was measured (Table 5
; Brito et al., 1997
). This activity was reduced sixfold in the IHF mutant strain.
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Promoter P3 is not required for hydrogenase activity in R. leguminosarum pea bacteroids
To investigate the physiological relevance of P3 on hydrogenase expression in R. leguminosarum, strain UPM791P3, incorporating the mutation in the P3 54-binding sequence, was used. Both wild-type and mutant strains were used as inocula for pea plants and hydrogenase activity of bacteroids was determined. No significant differences in the level of O2-dependent hydrogenase activity from wild-type strain UPM791 and the mutant strain UPM791P3 were found [4290±360 vs 4230±620 nmol H2 oxidized h-1 (mg protein)-1]. A dot-blot analysis confirmed the same level of hupG mRNA signal in wild-type and UPM791P3 mutant strains (data not shown). These results indicate that P3 is not essential for R. leguminosarum hydrogenase activity in pea nodules under the experimental conditions tested. Since the gene products of hupGHIJ are essential for hydrogenase activity (Rey et al., 1992
; Brito et al., 1994
), this result also implies the existence of transcription originated upstream of P3, probably from the P1 promoter.
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DISCUSSION |
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The P3 promoter was activated by NifA from K. pneumoniae both in K. pneumoniae and in E. coli. In K. pneumoniae it was also activated by NtrC or other uncharacterized regulators, since a nifA mutation affected P3 expression less drastically than a ntrC mutation. The high levels of P3 activity obtained in E. coli or K. pneumoniae in the presence of extra copies of nifA allowed further characterization of the promoter. No binding sequences located upstream of the 54-binding box, either UASs or IHF-binding sites, were required for P3 activation, suggesting that P3 is activated by NifA from solution by direct interaction with the P3-bound
54-RNA polymerase. Instances of promoter activation by NifA from solution have been reported. The nifLA operon from K. pneumoniae is activated by high concentrations of NifA even though the nifL promoter lacks the conserved UAS postulated as the target for NifA (Drummond et al., 1983
). The removal of UASs from the S. meliloti nifH promoter results in normal expression in alfalfa nodule bacteroids (Better et al., 1985
). Similarly, the R. leguminosarum glnB gene promoter is activated by NtrC in the absence of UASs (Chiurazzi & Iaccarino, 1990
).
The above data are also consistent with the observed lack of activator specificity (Pérez-Martín & de Lorenzo, 1995) and imply that P3 can be activated from solution by any 54-RNA polymerase activator present.
The same results were obtained for symbiotic expression of P3 in R. leguminosarum. However, in view of the observed pattern of P3 expression in the nodule and of the fact that the ntrC gene is not expressed in pea bacteroids (Szeto et al., 1987), it is likely that the observed symbiotic activation of P3 is mediated by NifA.
The specific function of hupGHIJ gene products is unknown, but the need of these proteins for hydrogenase activity in R. leguminosarum has been established (Rey et al., 1992; Brito et al., 1994
). Genes homologous to hupGHIJ are also present in the hydrogenase gene cluster from other nitrogen-fixing bacteria such as Azotobacter vinelandii and B. japonicum, and in Ralstonia eutropha and Rhodobacter capsulatus (Casalot & Rousset, 2001
). A hupFhupG intergenic space long enough to accommodate a promoter similar to P3, and containing a conserved
54-binding sequence at the expected distance from hupG, has only been found in the endosymbiotic bacterium B. japonicum (Van Soom et al., 1993a
; Fu & Maier, 1994
). Symbiotic expression of the hupSL promoter is activated by FixK2 in this bacterium (Durmowicz & Maier, 1998
), and the presence of a NifA-dependent promoter activity upstream of hupG has not been investigated. It is unlikely that FixK2 would be the activator of a putative P3-like promoter, since it is an activator of
70-dependent-type promoters. In Ralstonia eutropha, no promoter region has been identified upstream of the hupGHIJ-homologous genes (hoxOQRT), and transcription originating from the membrane-bound hydrogenase structural gene promoter has been demonstrated (Schwartz et al., 1999
).
Although the hupGHIJ genes are preceded by the symbiotically functional P3, this promoter does not seem to be essential for their expression in the mature bacteroid within the nodule, as shown by the results obtained with the 54-box mutant UPM791P3. Transcription from P1, the hydrogenase structural gene promoter, is probably responsible for the expression of these genes in the UPM791P3 strain. These results suggest that the P3 promoter would only be relevant for expression of accessory genes hupGHIJ under conditions where transcription originating from the upstream promoter P1 becomes limiting for hydrogenase synthesis. Since transcription of the hupGHIJ genes from the P1 promoter produces transcripts of over 7 kb in length, any condition affecting the levels of these transcripts (diminished processivity of RNA polymerase, increased mRNA degradation) or their translation (ribosome processivity) would probably have a more severe effect on distal genes. Irrespective of what these conditions may be, it is noteworthy that the unique molecular adaptation of the hydrogenase gene cluster of Rhizobium leguminosarum UPM791 to symbiotic expression within the bacteroid not only includes the NifA-dependent expression of the P1 hydrogenase structural gene promoter (Brito et al., 1997
) but also that of the internal, secondary P3 promoter.
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ACKNOWLEDGEMENTS |
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Received 3 July 2003;
revised 12 November 2003;
accepted 18 November 2003.
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