Expression of the nifA gene of Herbaspirillum seropedicae: role of the NtrC and NifA binding sites and of the -24/-12 promoter element

E. M. Souzaa,1, F. O. Pedrosa2, L. U. Rigo2, H. B. Machado3 and M. G. Yatesa,1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The nifA promoter of Herbaspirillum seropedicae contains potential NtrC, NifA and IHF binding sites together with a -12/-24 {sigma}N-dependent promoter. This region has now been investigated by deletion mutagenesis for the effect of NtrC and NifA on the expression of a nifA::lacZ fusion. A 5’ end to the RNA was identified at position 641, 12 bp downstream from the -12/-24 promoter. Footprinting experiments showed that the G residues at positions -26 and -9 are hypermethylated, and that the region from -10 to +10 is partially melted under nitrogen-fixing conditions, confirming that this is the active nifA promoter. In H. seropedicae nifA expression from the {sigma}N-dependent promoter is repressed by fixed nitrogen but not by oxygen and is probably activated by the NtrC protein. NifA protein is apparently not essential for nifA expression but it can still bind the NifA upstream activating sequence.

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Herbaspirillum seropedicae is an endophytic nitrogen-fixing bacterium found associated with several grasses, which fixes nitrogen under micro-oxic conditions and in the absence of a fixed nitrogen source (Baldani et al., 1986 ; Boddey et al., 1995 ). Initially classified as an Azospirillum species, it is now placed in the rRNA superfamily III or ß-subdivision of the Proteobacteria (Young, 1992 ). This subdivision, which is the least studied genetically of nitrogen-fixing families, includes species of Rhodocyclus, Alcaligenes, Derxia and Azoarcus.

The NifA protein is a specific activator of the expression of nif gene promoters by interaction with {sigma}N ({sigma}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 {sigma}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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and plasmids.
The bacterial strains and plasmids used are listed in Table 1. The Herbaspirillum seropedicae strain SmR1 is a spontaneous streptomycin-resistant (100 µg ml-1) mutant isolated from H. seropedicae Z78 (Baldani et al., 1986 ).


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Table 1. Strains and plasmids

 
Media and growth conditions.
H. seropedicae was grown in JNFbP medium (Baldani et al., 1986 ) supplemented with 20 mM NH4Cl (JNFbPN). Escherichia coli was grown in LB or 2xYT medium (Sambrook et al., 1989 ). The minimal medium for E. coli was NFDM (Dixon et al., 1977 ) supplemented with 20 mM NH4Cl. The concentrations of antibiotics used were as follows: carbenicillin, 200 µg ml-1; tetracycline, 10 µg ml-1; streptomycin, 20 µg ml-1 (E. coli) or 100 µg ml-1 (H. seropedicae); kanamycin, 50 µg ml-1 (E. coli) or 250 µg ml-1 (H. seropedicae); chloramphenicol, 30 µg ml-1 (E. coli) or 150 µg ml-1 (H. seropedicae); nalidixic acid, 20 µg ml-1.

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 BamHI–HindIII fragment from pEMS101 containing H. seropedicae DNA was cloned in pSUP202, producing pEMS108. This plasmid was mutagenized using the Tn5lacZ construct Tn5–B21 (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 1–5% 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 PstI–XhoI fragment from plasmid pEMS109 (nifA::Tn5–B21), 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 EcoRI–NsiI, the 0·5 kb EcoRI–DraI and the 0·5 kb EcoRI–SspI subfragments from the 1·7 kb EcoRI fragment into pPW452 producing pEMS121, pEMS122 and pEMS123, and the 0·7 kb EcoRI–PmlI fragment into pMP220 yielding pEMS124. The 1·3 kb EcoRI–NsiI and 1·2 kb EcoRI–DraI fragments were cloned into pMP220 to produce pEMS125 and pEMS126, and the 1·0 kb EcoRI–PmlI fragment into pPW452 to produce pEMS127. The EcoRI–DraI and EcoRI–SspI fragments were cloned first into pTZ18 digested with EcoRI/SmaI and then transferred as EcoRI–PstI fragments to pMP220 and pPW452 digested with EcoRI/PstI (Table 1). The EcoRI–PmlI fragments were also cloned into pTZ18 digested with EcoRI/SmaI and cloned as EcoRI–BamHI fragments into pPW452 and pMP220 digested with EcoRI/BglII. The EcoRI–NsiI fragments were cloned in pMP220 and pPW452 digested with EcoRI/PstI (see Table 1).



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Fig. 1. Construction of nifA::lacZ fusions. (a) Nucleotide sequence of the promoter region of H. seropedicae nifA. The NtrC and NifA UASs are underlined, the -24/-12 promoter element is double underlined and the arrow indicates the RNA start site. (b) Scheme of the nifA::lacZ fusions. Restriction enzymes are: E, EcoRI; P, PmlI; S, SspI; D, DraI; N, NsiI.

 
ß-Galactosidase activity.
Studies of the expression of the H. seropedicae nifA gene in E. coli were as follows: NFDM (2 ml) in a bijou bottle containing glutamine (100 µg ml-1) plus antibiotics was inoculated with 50 µl of an overnight culture in 2xYT in air and shaken for 18–20 h at 30 °C. NH4Cl (20 mM) was added when necessary.

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 5–6 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 [{gamma}-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 0·5–0·7. 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) .


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction of nifA mutants of H. seropedicae
Plasmid pEMS109.1 was used to produce the mutant strain SmR54 that carries a kanamycin-resistance cassette inserted into the coding region of the nifA gene corresponding to the central domain of the NifA protein. This strain failed to reduce acetylene and was complemented by the plasmids pEMS107 (which expresses H. seropedicae nifA from the nifA promoter) and pEMS135 (which expresses nifA constitutively from the lac promoter). pEMS109 was used to insert Tn5–B21 in the H. seropedicae nifA gene, producing strain SmR2532 carrying the lacZ gene in the same orientation as the nifA gene. This strain was also Nif- and was complemented by plasmid pEMS101, which carries the nifA gene from H. seropedicae.

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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Table 2. ß-Galactosidase activity of nifA::lacZ fusions in E. coli

 
When a nifA::lacZ fusion was cloned into a medium-copy-number vector (pBR322) however, the effect of the NtrC binding site was clearer: deletion of the NtrC UAS in pEMS114 reduced activity fivefold compared to that of pEMS111 (Table 2). In E. coli ET8045 (rpoN-) neither NifA nor NtrC activated the nifA fusion carried by pEMS109 whereas in ET8894 (rpoN+) a 10-fold or 20-fold increase in ß-galactosidase activity was observed when NifA or NtrC, respectively, was expressed constitutively (Table 2). This result indicates that the nifA promoter is dependent on the presence of the rpoN gene product. Addition of NH4Cl (20 mM) did not alter nifA expression in E. coli (not shown).

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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Table 3. ß-Galactosidase activity of nifA::lacZ fusions in H. seropedicae

 
Expression of plasmid-borne nifA::lacZ fusions in H. seropedicae.
As in the chromosomal mutant, the ß-galactosidase expression of the fusion carried by plasmid pEMS120 (comprising the whole promoter region of the nifA gene) in H. seropedicae strain SmR1 was also depressed 4–5-fold in the presence of 20 mM NH4Cl but was not substantially affected by oxygen (Table 3). Similar levels of expression were observed in strain SmR54 (nifA mutant) carrying pEMS120. Deletion of the sequence upstream from base 264 (pEMS124), including half of the NtrC binding site, decreased the ß-galactosidase activity in SmR1 under normal N2-fixing conditions by 50%; this activity was only depressed 30% by ammonia. In strain SmR54, expression from this mutant promoter was lower under N2-fixing conditions than in the wild-type (SmR1) with the same deletion and it was insensitive to either added ammonium or air but was depressed further by 50% when both were added together. Deletion of 179 bp (pEMS123) and 190 bp (pEMS122) from base 264 caused an increase in ß-galactosidase activity under derepressing conditions and marked repression by either ammonium ions or oxygen in the wild-type strain, while in the nifA mutant there was no response to the absence of fixed nitrogen and low oxygen. The ß-galactosidase activity of pEMS122 was twice that of pEMS123 in the derepressing wild-type strain.

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 3–4-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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Fig. 2. Mapping of the transcriptional-start site of the nifA gene of H. seropedicae. The lanes labelled 5U and 50U show the S1 nuclease digestion products with 5 and 50 units of S1 nuclease, respectively, from the hybridization reaction between total RNA and the single-stranded probe synthesized using primer 1 (Methods). The arrow indicates the transcription-start site.

 
In vivo footprinting
In vivo footprinting in E. coli.
To determine if NifA could bind to the NifA UAS (position 504 to 519) in the promoter region of the H. seropedicae nifA, the plasmids pEMS111 (carrying the nifA promoter) and pNH11 (expressing K. pneumoniae nifA from the tac promoter) were transformed into E. coli and the culture treated with DMS. In this experiment, the binding of the protein to the DNA is assessed by a relative decrease in methylation (protection) of the G residues and/or by an enhancement of methylation if the base is more exposed to the chemical modification upon protein binding. The G residue of the TGT motif at positions 505 and 518 of the top and bottom strands, respectively, were strongly protected in the presence of K. pneumoniae NifA (Fig. 3a, b, lanes 4 and 5), indicating NifA binding to the UAS in vivo. The residues at positions 522 and 525 of the top strand and 527 of the bottom strand were weakly protected by K. pneumoniae NifA. These residues are outside of the NifA UAS but the sequence 522-GACGACA may constitute a half NifA UAS.



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Fig. 3. Interaction of NifA UAS of H. seropedicae nifA promoter with the NifA protein. Bands protected from methylation by DMS in the presence of NifA expressed constitutively in E. coli or under nitrogen-fixing conditions in H. seropedicae are indicated by {circ}. (a) Top strand. (b) Bottom strand. Lanes: 1, SmR1(pEMS120) under nitrogen-fixing conditions; 2, SmR1(pEMS120) plus 20 mM NH4Cl; 3, SmR54(pEMS120) under nitrogen-fixing conditions; 4, ET8894(pEMS111) with K. pneumoniae NifA provided by pNH11; 5, ET8894(pEMS111).

 
In vivo footprinting in H. seropedicae.
To identify the interaction of the -24/-12 promoter sequence with the {sigma}N-containing RNA polymerase, cultures of H. seropedicae harbouring pEMS120 in the absence or presence of NH4Cl with and without rifampicin, were chemically treated with DMS or permanganate. In the DMS footprinting, methylation of the residue at position 615 (-26) of the top strand was enhanced in the absence of fixed nitrogen and that of residue 632 (-9) when rifampicin was also added (Figs 4 and 5). In the bottom strand there was no substantial difference between the four conditions (not shown). Enhancement of methylation of residue -26 might reflect a higher occupancy of the promoter by {sigma}N-containing RNA polymerase under nitrogen-fixing conditions, whereas the altered reactivity of residue -9 might be due to formation of an open complex. DMS footprinting of the promoter region of the nifA in an E. coli rpoN+ (ET8894) strain revealed that the G residue at position 616 (-25) in the top strand was protected in the presence of the rpoN gene (not shown), also suggesting interaction of the {sigma}N-containing RNA polymerase with this sequence. In the bottom strand no protection was observed.



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Fig. 4. DMS footprinting of the top strand of the -24/-12 element of H. seropedicae nifA. Hypermethylation is indicated by {bullet}. Lanes: 1, SmR1(pEMS120) under nitrogen-fixing conditions; 2, SmR1(pEMS120) plus 20 mM NH4Cl; 3, SmR1(pEMS120) under nitrogen-fixing conditions plus rifampicin; 4, SmR1(pEMS120) plus 20 mM NH4Cl and rifampicin. Rifampicin was added to block RNA synthesis and trap the nifA promoter in the open-complex form. For comparison, the C sequencing track (corresponding to the G position of the top strand) is shown. No obvious protection was detected.

 


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Fig. 5. Densitometric analysis of the DMS footprint at top strand of the H. seropedicae nifA promoter. (a) SmR1(pEMS120) under nitrogen-fixing conditions. (b) SmR1(pEMS120) plus 20 mM NH4Cl. (c) SmR1(pEMS120) under nitrogen-fixing conditions plus rifampicin. (d) SmR1(pEMS120) plus 20 mM NH4Cl and rifampicin. (e) The autoradiographs were scanned and the peak heights determined. The normalized height of each peak under nitrogen-fixing conditions was divided by the value in the presence 20 mM NH4Cl and expressed as the logarithm of the quotient. Peaks were normalized against the value of residues 598. Rifampicin was added (black bars) or not (white bars) to trap open complexes. Positive values indicate hypermethylation and negative values indicate protection. Guanine residue -26 is hyper-reactive under derepressing conditions while residue -9 is hypermethylated only when rifampicin was added to derepressed cells.

 
Permanganate footprinting probes for open-complex formation, since T residues of single-stranded DNA become more reactive. The T residue at position 650 (+10) in the top strand was slightly more reactive towards permanganate in the absence of ammonium ions than in their presence (Fig. 6a, lanes 3 and 4; Fig. 7). When rifampicin was added to trap open complexes, T residues at positions 631, 643, 647, 649 and 650 (-10, +3, +7, +9 and +10) were more reactive in the absence than in the presence of fixed nitrogen (Fig. 6a, lanes 1 and 2; Fig. 7). These results suggest melting of the region between residues -10 and +10 during open-complex formation under nitrogen-fixing conditions. The only residue in the bottom strand to show an increased reactivity towards permanganate was T 633 (position -8) in the presence of rifampicin (Fig. 6b). Permanganate footprinting of the nifA promoter was determined in ET8894 (rpoN+) and ET8045 (rpoN-) carrying pEMS109 (HsnifA promoter) and pCK1 (K. pneumoniae nifAc) in the presence of rifampicin. In the rpoN+ strain, the residues at positions 631, 643 and 647 in the top strand (-10, +3 and +7 in relation to the 5' end of nifA RNA) showed an increase in reactivity towards KMnO4 (not shown). No increase in reactivity was identified in the bottom strand.



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Fig. 6. Probing open complexes at the H. seropedicae nifA promoter with KMnO4. (a) Top strand. (b) Bottom strand. Lanes: 1, SmR1(pEMS120) under nitrogen-fixing conditions plus rifampicin; 2, SmR1(pEMS120) plus 20 mM NH4Cl and rifampicin; 3, SmR1(pEMS120) under nitrogen-fixing conditions; 4, SmR1(pEMS120) plus 20 mM NH4Cl. Rifampicin was added to block RNA synthesis and trap the nifA promoter in the open complex form. For comparison, the A sequencing track (corresponding to the T positions) is shown.

 


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Fig. 7. Densitometric analysis of potassium permanganate footprinting of the top strand at the H. seropedicae nifA promoter. (a) SmR1(pEMS120) under nitrogen-fixing conditions plus rifampicin. (b) SmR1(pEMS120) plus 20 mM NH4Cl and rifampicin. (c) SmR1(pEMS120) under nitrogen-fixing conditions. (d) SmR1(pEMS120) plus 20 mM NH4Cl. (e) The autoradiographs were scanned and the peak heights determined. The normalized height of each peak under nitrogen-fixing conditions was divided by the value in the presence of 20 mM NH4Cl and expressed as the logarithm of the quotient. Peaks were normalized against the value of residues 610. Rifampicin was added (black bars) or not (white bars) to trap open complexes. Positive values indicate hyper-reactivity towards permanganate.

 
To determine whether there was a change in methylation by DMS in the NifA UAS (positions 504–519), plasmid DNA was purified from H. seropedicae strain SmR1 (pEMS120) derepressed in either the absence or presence of 20 mM NH4Cl and from H. seropedicae strain SmR54 (pEMS120) derepressed in the absence of fixed nitrogen. The G residue at position 505 of the top strand and the residue 518 of the bottom strand were protected from methylation by DMS in the absence of NH4Cl, indicating binding of the NifA protein under nitrogen-fixing conditions (Fig. 3a, b, compare lane 1 with lanes 2 and 3).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results of the expression of the nifA gene of H. seropedicae in an enteric background (Table 2) indicated that the transcription of this gene was activated by either the NifA or NtrC proteins and is dependent on the {sigma}N factor. Indeed, the nifA RNA 5' end was mapped under derepressing conditions at position 641 and, 11 bp upstream from this position, a sequence homologous to a -24/-12 promoter element (nifAp) is located (Fig. 1a). The relatively high activity of the fusion with the NtrC UAS deleted may be due to interaction between non-bound NtrC and the closed promoter/{sigma}N-containing RNA polymerase complex.

The expression of nifA in H. seropedicae is regulated by fixed nitrogen (4–5-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 522–528 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 {sigma}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 {sigma}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 {sigma}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.


   ACKNOWLEDGEMENTS
 
We thank Martin Buck for discussion and suggestions, and Rose A. Monteiro and Fabiane M. Rego for assistance with the densitometric analysis. We also thank CNPq and FINEP/MCT/Pronex for financial support.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 6 July 1999; revised 29 December 1999; accepted 21 March 2000.