BBSRC, IPSR Nitrogen Fixation Laboratory, University of Norwich, Norwich, UK1
Departamento de Bioquímica UFPR, C. Postal 19046, 81531-970, Curitiba, PR, Brazil2
Departamento de Farmacologia, UFPR, 81531-990, Curitiba, PR, Brazil3
Author for correspondence: E. M. Souza. Tel: +55 41 266 2042. Fax: +55 41 366 4398. e-mail: souzaem{at}bio.ufpr.br
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ABSTRACT |
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Keywords: Herbaspirillum seropedicae, nitrogen fixation, nifA gene
Abbreviations: DMS, dimethysulphate; UAS, upstream activating sequence
a Present address: Departamento de Bioquímica UFPR, C. Postal 19046, 81531-970, Curitiba, PR, Brazil.
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INTRODUCTION |
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The NifA protein is a specific activator of the expression of nif gene promoters by interaction with N (
54, RpoN)-containing RNA polymerase, which recognizes promoters with the dinucleotides GG and GC conserved at positions -25/-24 and -12/-13, respectively (Dixon, 1988
). The NifA protein binds to a specific upstream activating sequence (UAS) and interacts with the
N-containing RNA polymerase bound to the promoter to induce open complex formation (Morett & Buck, 1989
). This mechanism is common to all nitrogen-fixing proteobacteria to date.
The regulation of nifA expression, on the other hand, differs, depending on the organism. In Klebsiella pneumoniae, the phosphorylated NtrC protein activates nifA gene expression under nitrogen limitation. NtrC is phosphorylated by NtrB, whose activity is controlled by the cellular nitrogen status via the proteins GlnB and GlnD (see Merrick & Edwards, 1995 ). In Rhizobium meliloti (now Sinorhizobium meliloti), nifA and fixK expression (Batut et al., 1989
; David et al., 1988
) is activated by the FixJ protein, which is inactivated under high oxygen by the FixL protein, while the FixK protein negatively regulates nifA expression. The NifA protein also activates its own expression (Batut et al., 1989
). In Azorhizobium caulinodans full expression of the nifA promoter occurs only under micro-oxic and low fixed nitrogen conditions (Loroch et al., 1995
) when FixJ activates fixK expression and FixK, in turn, activates nifA expression, in apparent contrast to the situation in S. meliloti. A NifA UAS is also present in the A. caulinodans nifA promoter and is possibly involved in negative auto-regulation (Nees et al., 1988
; Stigter et al., 1992
). Finally, the NrfA protein has also been implicated in translational nifA regulation (Kaminski & Elmerich, 1998
). The nifA gene of Bradyrhizobium japonicum is in the operon fixRnifA, which is positively regulated by an oxygen-sensitive NifA protein (Fischer & Hennecke, 1987
). In this organism, however, nifA is expressed primarily from another promoter activated by the RegR protein (Bauer et al., 1998
). The nifA gene expression in Azospirillum brasilense is not tightly regulated by either fixed nitrogen or oxygen, although a significant decrease of expression occurs in the presence of high levels of both ammonia and oxygen together (Liang et al., 1991
). Both ammonia and oxygen, on the other hand, apparently regulate NifA activity since there is no activation of nifH under such conditions. The PII protein probably participates in the regulation of NifA protein activity since a glnB null mutant of A. brasilense is Nif- even when the nifA gene is expressed constitutively (de Zamaroczy et al., 1993
; Arsene et al., 1996
).
The promoter region of the nifA gene of H. seropedicae was found to contain sequences homologous to a -24/-12 type promoter, together with NifA and NtrC binding sites (Souza et al., 1991a ), suggesting participation of these proteins in the expression of the nifA gene. The activity of the NifA protein from H. seropedicae is controlled by ammonium ions and oxygen (Souza et al., 1999
). nif gene expression in H. seropedicae apparently requires a functional glnB-like gene (Benelli et al., 1997
).
Here we report the identification of a major transcriptional-start site of the nifA gene of H. seropedicae under nitrogen-fixing conditions, verification of a -24/-12 transcriptionally active promoter and an analysis of the roles of NifA and NtrC proteins in the activation of nifA expression.
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METHODS |
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Conjugation.
Plasmids were introduced into H. seropedicae by triparental matings (Pedrosa & Yates, 1984 ), using pRK2013 as the helper plasmid.
Construction of nifA mutants.
A 6·5 kb BamHIHindIII fragment from pEMS101 containing H. seropedicae DNA was cloned in pSUP202, producing pEMS108. This plasmid was mutagenized using the Tn5lacZ construct Tn5B21 (Simon et al., 1989 ) to obtain a transposon insertion approximately 300 bp downstream from the nifA translation start. The orientation of lacZ in this plasmid, named pEMS109, was the same as that of the nifA gene. A 2 kb SalI fragment from pEMS101, containing part of the nifA gene, was also cloned into pSUP202 and the kanamycin-resistance cassette from pKIXX was inserted into the BglII site in the nifA gene to give the plasmid pEMS109.1. H. seropedicae nifA mutants were then obtained by introducing these mutated plasmids pEMS109 and pEMS109.1 into H. seropedicae by conjugation, producing strains SmR2532 and SmR54, respectively. Recombinant colonies resistant to the transposon or cassette markers were screened for a combination of the vector marker (chloramphenicol sensitivity), ß-galactosidase and nitrogenase activities. Only 15% of the recombinant colonies were products of a double crossover, as judged by the above parameters. The transposon or cassette insertion in the nifA gene was confirmed by chromosomal DNA hybridization following restriction and electrophoresis (not shown).
Construction of nifA::lacZ fusions.
The 6·5 kb PstIXhoI fragment from plasmid pEMS109 (nifA::Tn5B21), containing the whole nifA promoter region and the lacZ gene, was cloned into EcoRI/PstI-digested pBR322, yielding pEMS111. pEMS114 was constructed by cutting pEMS111 with DraI and religating. This construction contains the NifA UAS and the -24/-12 promoter sequence.
Another set of nifA::lacZ fusions was constructed using the IncP fusion vectors pPW452 and pMP220 (Fig. 1a, b
). A 1·7 kb EcoRI fragment containing part of the N-terminal of the nifA gene and the whole promoter region was cloned into the transcriptional fusion vector pPW452, yielding the plasmid pEMS120, in which lacZ expression was under control of the nifA promoter. Deletions in the promoter region were prepared by cloning the 0·4 kb EcoRINsiI, the 0·5 kb EcoRIDraI and the 0·5 kb EcoRISspI subfragments from the 1·7 kb EcoRI fragment into pPW452 producing pEMS121, pEMS122 and pEMS123, and the 0·7 kb EcoRIPmlI fragment into pMP220 yielding pEMS124. The 1·3 kb EcoRINsiI and 1·2 kb EcoRIDraI fragments were cloned into pMP220 to produce pEMS125 and pEMS126, and the 1·0 kb EcoRIPmlI fragment into pPW452 to produce pEMS127. The EcoRIDraI and EcoRISspI fragments were cloned first into pTZ18 digested with EcoRI/SmaI and then transferred as EcoRIPstI fragments to pMP220 and pPW452 digested with EcoRI/PstI (Table 1
). The EcoRIPmlI fragments were also cloned into pTZ18 digested with EcoRI/SmaI and cloned as EcoRIBamHI fragments into pPW452 and pMP220 digested with EcoRI/BglII. The EcoRINsiI fragments were cloned in pMP220 and pPW452 digested with EcoRI/PstI (see Table 1
).
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Derepression experiments in H. seropedicae were conducted with overnight-grown cultures. The cells were collected by centrifugation and resuspended in either fresh JNFbP or JNFbPN to an OD600 of 0·2. The cell suspension (2 ml) was transferred to 25 ml conical flasks containing either oxygen (1·5% in N2) or air and shaken for 56 h at 30 °C. The ß-galactosidase activity was then determined, using ONPG as the substrate (Miller, 1972 ).
RNA 5 end mapping.
RNA was purified from H. seropedicae grown under nitrogen-fixing conditions (Krol et al., 1982 ). The single-stranded probe used for S1 mapping was obtained by asymmetric PCR using 2 pmol primer 1 (5'-ATGCGCTGCCTGAGAGCGCT-3') end-labelled with [
-32P]ATP (Amersham). Conditions of hybridization and digestion were as in Sambrook et al. (1989)
.
Recombinant DNA techniques.
All DNA manipulations including cloning, end-labelling and transformations were performed by current techniques (Sambrook et al., 1989 ).
In vivo footprinting.
For dimethylsulphate (DMS) footprinting, an overnight culture of E. coli ET8894 carrying the relevant plasmids was diluted to an OD600 of 0·1 in 2xYT and shaken at 30 °C until the OD600 was 0·50·7. The culture was treated with DMS (0·05%, v/v) for 2 min; the cells were then collected by centrifugation and washed in Tris/HCl (50 mM, pH 8·0), EDTA (10 mM) solution. The methylated plasmids were purified by alkaline lysis (Sambrook et al., 1989 ).
For in vivo DMS footprinting in H. seropedicae, an overnight culture of a strain carrying pEMS120 (nifA::lacZ fusion) was centrifuged, diluted to an OD600 of 1·1 in 20 ml JNFbP or JNFbPN in a 65 ml bottle and incubated in a rotary shaker at 120 r.p.m. for 5 h. The culture was treated with freshly prepared DMS (0·05%, v/v, final concn) for 2 min and plasmid purification was done as above. When used, rifampicin (100 µg ml-1) was added 7 min before the addition of DMS. To cleave the DNA at the methylated G residues, the plasmid DNA isolated after DMS treatment was dissolved in 30 µl 1 M piperidine, incubated at 90 °C for 30 min and submitted to 3 cycles of lyophilization followed by dissolution in 20 µl water. The cleaved DNA was used as the template for primer extension with Taq DNA polymerase (Sasse-Dwight & Gralla, 1991 ).
For in vivo KMnO4 footprinting, an overnight culture of a H. seropedicae strain carrying the nifA::lacZ fusion was derepressed as above. Freshly prepared potassium permanganate (15 mM, final concn) was added and the cultures incubated for 5 min. When required, rifampicin (100 µg ml-1) was added to trap open complexes 7 min before addition of KMnO4. Plasmid purification, cleavage with piperidine and extension was as above.
Autoradiograms were analysed densitometrically in a Molecular Dynamics PSI Densitometer and differences in the level of band reactivity were calculated as described by Moret & Buck (1988) .
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RESULTS |
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Expression of the H. seropedicae nifA gene in E. coli
The nifA::lacZ fusion in plasmid pEMS120 carrying the whole promoter region of the nifA gene of H. seropedicae in the low-copy-number vector pPW452 exhibited a very low expression rate in E. coli ET8894, which increased 50-fold when the NifA protein from K. pneumoniae was provided by the plasmid pMC71A (Table 2). Deletion of the NifA UAS (pEMS121) reduced the activity of the nifA promoter by 90% in the presence of K. pneumoniae NifA. Deletion of DNA sequences upstream from base 453 (pEMS122) resulted in a twofold increase in the NifA-dependent ß-galactosidase activity. The NtrC protein of K. pneumoniae expressed from pMM14 also activated expression of the nifA gene (pEMS120). This activation was reduced (65%) when the NtrC binding site was completely (pEMS122) or partially (pEMS124) deleted.
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Expression of the nifA gene in H. seropedicae
Expression of the chromosomal nifA gene in H. seropedicae nifA::lacZ.
Fixed nitrogen (20 mM NH4Cl) decreased the expression of the H. seropedicae chromosomal nifA::lacZ fusion (strain SmR2532) by 80%, but oxygen did not repress expression (Table 3).
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Finally, elimination of a region upstream from position 604 (pEMS121), leaving only the -24/-12 promoter sequence, produced a fusion with a low level of expression not regulated by either ammonium ions or oxygen (Table 3). Clearly, the upstream sequences are important for promoter activity.
A set of progressive deletions from the 3' end of the nifA promoter was also tested. Deleting the -24/-12 promoter (pEMS125) caused a large decrease of ß-galactosidase activity under all conditions. With pEMS126, where both the NifA UAS and the promoter region were eliminated, there was a weak non-regulated expression. That level was increased 34-fold in pEMS127, where a further 220 bp were deleted (Table 3).
Mapping of the 5' end of nifA RNA
To identify the 5' end of the nifA RNA, a single-stranded probe was synthesized by unidirectional PCR using an oligonucleotide primer labelled with 32P. This probe was used for S1 mapping, revealing position 641 as the nifA RNA 5' end (Fig. 2). No signal was detected with RNA isolated from cultures under repressing conditions (20 mM NH4Cl; not shown).
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DISCUSSION |
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The expression of nifA in H. seropedicae is regulated by fixed nitrogen (45-fold reduction by NH4Cl) but not by oxygen (Table 3). The expression levels of nifA in the H. seropedicae nifA mutant carrying pEMS120 were similar to those in the wild-type carrying the same plasmid, which suggests that, contrary to results in E. coli, the NifA protein is not essential for the expression of the nifA gene in H. seropedicae. Partial deletion of the NtrC UAS (pEMS124) abolished the regulation by ammonium ions but decreased by half the ß-galactosidase activity in the wild-type carrying pEMS120, indicating that the putative NtrC UAS is necessary for activation of nifA gene expression in H. seropedicae. These results, together with transcription-start-site mapping, indicate that the increase in nifA expression under nitrogen-fixing conditions is primarily dependent on the NtrC activation of the -24/-12 promoter element.
Regulation by ammonium or oxygen, as well as dependence upon the NifA protein, was introduced when all but 70 bp upstream of the putative NifA binding site (pEMS123, Fig. 1b) was deleted. Elimination of a further 20 bp (pEMS122) had the same pattern, although the ß-galactosidase activity under nitrogen-fixing conditions was higher in the wild-type strain carrying pEMS122 than that carrying pEMS123 (Table 3
). Interestingly, the fusion carried by pEMS122 expressed ß-galactosidase at twice the level of that of pEMS120, suggesting that the sequences upstream from the NifA binding site interfere with the ability of NifA to activate this promoter.
Although NifA is not essential for nifA expression (579 Miller units in the wild-type compared to 418 in the NifA- background), it may contribute to the full expression at the promoter. Moreover, the ß-galactosidase activities with the deletions in plasmids pEMS123 and pEMS122 indicate that the NifA UAS is functional under certain conditions in H. seropedicae. This conclusion is also supported by footprinting evidence showing that NifA does interact with its putative UAS. DMS footprinting in E. coli showed that the G residues of TGT motifs in both top and bottom strands were strongly protected by NifA (Fig. 3a, b
); two positions in the NifA UAS of K. pneumoniae nif genes that are characteristically protected by bound NifA (Morett & Buck, 1988
). The half NifA UAS located at position 522528 was also protected, possibly due to the high concentration of the NifA protein produced by pNH11 allowing for the binding to a lower-affinity DNA sequence. In H. seropedicae the G residues of the TGT motif of the NifA UAS were protected in both strands in the absence of fixed nitrogen. Since nifA expression is activated under this condition, this result suggests that the NifA protein is able to bind to the NifA UAS in H. seropedicae. Plasmids pEMS122 and pEMS123, which lack the sequences at the 5' end of the NifA UAS, show higher activation dependent on NifA. It is possible that the sequences close to the 5' end of the NifA UAS play a regulatory role in NifA activation of the -24/-12 promoter. The sequence ~50 bp upstream from the NifA UAS may constitute an IHF binding site (Souza et al., 1991a
). Presumably the IHF could inhibit NifA binding and hence transcription in the absence of a NtrC UAS. This possibility parallels the observation by Austin et al. (1994)
that NifA-dependent in vitro transcription of the nifH promoter was inhibited by the IHF if the NifA UAS was deleted. IHF and NifA may compete for binding in this DNA region and regulate nifA expression by a feedback mechanism. Only when the NifA concentration is high enough can it out-compete the IHF and activate the -24/-12 promoter. Alternatively, a high concentration of NifA might activate the promoter with binding to the UAS.
DMS footprinting of the putative -24/-12 promoter of the nifA gene confirmed that this region was in contact with the N-containing RNA polymerase. The -25 G residue (in relation to the RNA 5' end) alone was protected in the top strand but no base was protected in the bottom strand in E. coli (not shown) indicating weak contact, since the residues -25, -24 and -12 are characteristically protected by
N-containing RNA polymerase in E. coli (Buck & Cannon, 1989
). In H. seropedicae, when the promoter was trapped in the open-complex form by the addition of rifampicin, residues -26 and -9 were hypermethylated under nitrogen-fixing conditions, suggesting contact with the
N-containing RNA polymerase. Hypermethylation of residue -9 by DMS also occurs in the open complex of the nifU promoter of K. pneumoniae (Buck & Cannon, 1992
). Finally, the permanganate footprinting (Fig. 6
) showed that the region between -10 and +10 of the H. seropedicae nifA promoter was more reactive towards permanganate, and probably engaged in an open complex under nitrogen-fixing conditions. These results indicated that the identified -24/-12 promoter sequence of H. seropedicae nifA is transcriptionally active in the absence of fixed nitrogen.
In pEMS127, where the -24/-12 promoter, the NifA and NtrC UASs were deleted, nifA expression was low and not regulated by ammonium ions or oxygen, neither in E. coli nor in H. seropedicae. In addition, a background level of nifA expression was present under repressing conditions, suggesting the presence of an upstream promoter. This promoter organization could maintain a constitutive low level of nifA RNA synthesis, allowing a low level of NifA protein to activate the expression of nif genes as soon as conditions become favourable for nitrogen fixation. On the other hand, smaller deletions of the 3' region, which leave the NtrC and NifA binding sites present, decreased the level of expression from this putative promoter in pEMS125 and pEMS126 in H. seropedicae. In E. coli pEMS125 also had a very low level of expression. It is possible that the region between bases 264 and 442 may destabilize the RNA or that binding of regulatory proteins diminishes transcription.
The nifA promoter active under nitrogen-fixing conditions is of the type -24/-12. This promoter is transcriptionally active in both E. coli and H. seropedicae. In H. seropedicae this promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein. NifA is apparently not essential for nifA expression but it can still bind the NifA UAS and may activate transcription when present in sufficient amounts to out-compete the IHF.
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ACKNOWLEDGEMENTS |
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Received 6 July 1999;
revised 29 December 1999;
accepted 21 March 2000.