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Negative Regulation of Expression of the Nitrate Assimilation

nirA Operon in the Heterocyst-Forming Cyanobacterium

Anabaena sp. Strain PCC 7120

José Enrique Frías and Enrique Flores*

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de

Investigaciones Científicas y Universidad de Sevilla, Centro de

Investigaciones Científicas Isla de la Cartuja, E-41092, Seville, Spain.

*Corresponding author. Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de

Investigaciones Científicas Isla de la Cartuja, Avenida Américo Vespucio 49, E-41092,

Seville, Spain. Tel.: +34 954489523; fax: +34 954460065.

E-mail address: eflores@ibvf.csic.es (E. Flores)

Running title: Regulation of the nirA operon

Keywords: Anabaena, cyanobacteria, nitrate assimilation, regulation

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01668-09 JB Accepts, published online ahead of print on 26 March 2010

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Abstract

In the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain

PCC 7120, expression of the nitrate assimilation nirA operon takes place in the

absence of ammonium and the presence of nitrate or nitrite. Several positive-

action proteins that are required for expression of the nitrate assimilation nirA

operon have been identified. Whereas NtcA and NtcB exert their action by direct

binding to the nirA operon promoter, CnaT acts by an as-yet-unknown

mechanism. In the genome of this cyanobacterium, ORF all0605 (the nirB gene) is

found between the nirA (encoding nitrite reductase) and ntcB genes. A nirB mutant

was able to grow at the expenses of nitrate as a nitrogen source and showed

abnormally high levels of nirA operon mRNA both in the presence and in the

absence of nitrate. This mutant showed increased nitrate reductase activity but

decreased nitrite reductase activity, an imbalance that resulted in excretion of

nitrite, which accumulated in the extracellular medium when the nirB mutant was

grown in the presence of nitrate. A deletion-in-frame nirA mutant also showed a

phenotype of increased expression of the nirA operon in the absence of ammonium

independent of the presence of nitrate in the medium. Both NirB and NirA are

therefore needed to keep low levels of expression of the nirA operon in the absence

of an inducer. Because NirB is also needed to attain high levels of nitrite reductase

activity, NirA appears to be a negative element in the nitrate-regulation of the

expression of the nirA operon in Anabaena sp. strain PCC 7120.

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Assimilatory nitrate reduction is carried out by many plants, algae, fungi and

bacteria. It involves the uptake of nitrate into the cell and its two-step reduction via

nitrite to ammonium, which is incorporated into carbon skeletons. In bacteria, uptake is

carried out by ABC-type or MFS transporters, and reduction involves the direct transfer

of electrons to nitrate and nitrite via nitrate reductase and nitrite reductase, respectively,

from iron-sulfur or flavin-containing donor proteins (28). Expression of the nitrate

assimilation system is frequently subjected to a dual regulation: repression by

ammonium and induction by nitrate (or nitrite). Whereas repression is exerted by the

general nitrogen control system of the bacterium, a variety of different mechanisms

appear to exist that mediate induction (28).

Cyanobacteria are photoautotrophs that carry out oxygenic photosynthesis.

Nitrate and ammonium are excellent sources of nitrogen for cyanobacteria in general,

and many strains are able to use urea or to fix atmospheric nitrogen (13, 15). In

cyanobacteria, reduction of nitrate to ammonium is catalyzed by two ferredoxin-

dependent enzymes, nitrate reductase and nitrite reductase. Genes encoding nitrite

reductase (nirA), an ABC-type nitrate/nitrite uptake transporter (nrtABCD), and nitrate

reductase (narB) are clustered together constituting the nirA operon (nirA-nrtABCD-

narB) in the genomes of Synechococcus elongatus strain PCC 7942 (hereafter referred

to as S. elongatus) and Anabaena sp. strain PCC 7120(13). Several genes involved in

the biosynthesis of the nitrate reductase molybdenum cofactor (molybdopterin guanine

dinucleotide) and two additional genes, narM and nirB, that affect nitrate reductase and

nitrite reductase activity levels, respectively, have also been identified in S. elongatus

(13). The nirB gene has been shown to be required for attaining maximum levels of

nitrite reductase, and its inactivation provokes an imbalance between nitrate and nitrite

reduction resulting in release of nitrite to the external medium (36).

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Nitrate reductase and nitrite reductase activities are lower in ammonium-grown

than in nitrate-grown cyanobacterial cells (13, 15). Expression of these enzyme

activities takes place at appreciable levels in the absence of nitrate or nitrite in some

cyanobacteria such as S. elongatus, but not in the heterocyst-forming, N2-fixing

cyanobacteria such as Anabaena sp. strain PCC 7120. Thus, in the non-N2-fixing

cyanobacteria the nitrate assimilation system is mainly subjected to ammonium-

promoted repression, whereas in the N2-fixing cyanobacteria, in addition to repression

by ammonium, induction by nitrate or nitrite is also required for attaining high levels of

expression, giving rise to a “nitrate effect” in this type of cyanobacteria (13, 15).

Expression of the nirA operon upon ammonium withdrawal is promoted by the

NtcA protein, a CAP-family transcription factor that is widespread among

cyanobacteria (25). NtcA activity is enhanced by 2-oxoglutarate, a putative C to N

balance signal in the cyanobacterial cell (17, 30) that can act on NtcA both directly (3,

33, 37-39) and indirectly via the signal-transduction protein PII (13, 33). In addition to

NtcA, a route-specific, LysR-type trancriptional regulator, NtcB, is involved in the

regulation of nirA operon expression (1, 2, 18, 27). In contrast to NtcA that is strictly

necessary for expression of the nirA operon in all investigated cyanobacterial strains,

NtcB is involved in regulation with different stringency levels depending on the

cyanobacterial strain. In the case of Anabaena sp. strain PCC 7120, the NtcB protein is

strictly required for expression of the nirA operon and for growth at the expense of

nitrate, and expression of ntcB itself takes place from an NtcA-dependent promoter (18).

A third positive regulatory element of nirA operon expression in Anabaena sp. strain

PCC 7120 is the CnaT protein (20), which shows overall sequence similarity to glycosyl

transferases. An Anabaena cnaT insertional mutant is unable to use nitrate as a nitrogen

source due to a defect in activation of transcription of the nirA operon. However, CnaT

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does not appear to be a DNA-binding protein and, consequently, the effect of CnaT on

nirA operon expression could be indirect.

Four open reading frames (ORFs all0603, all0604, all0605 and all0606) are

located in the genome of Anabaena sp. strain PCC 7120 between the nirA and ntcB

genes, with the same orientation as ntcB (Fig. 1). all0603 would encode a 101-amino

acid transcriptional regulator of the XRE-family. all0604 would encode a 119-amino

acid polypeptide that shows no homology with any protein of known function. all0605

(previously designated orf398 (19) encodes a protein with sequence similarity to several

proteins previously characterized in S. elongatus: the phycocyanobilin lyase alpha

subunit CpcE (26 % identity in a 163 amino acid overlap), the NblB polypeptide

involved in phycobilisome degradation (22 % identity in a N-terminal 180 amino acid

overlap plus 23 % identity in a C-terminal 148 amino acid overlap), and the NirB

protein (23 % identity in a 331 amino acid overlap). all0606 (previously designated

orf136 (19) would encode a protein similar to the cytochrome b6f-complex iron-sulfur

protein PetC. Besides all0606, three other Anabaena sp. strain PCC 7120 ORFs,

all2453, all4511, and all1512, show overall homology to the cytochrome b6f-complex

iron-sulfur protein PetC (26).

In this study, we show that, in addition to the positive elements described above,

the expression of the Anabaena nirA operon is subjected to the action of two negative

elements, the products of ORF all0605 (the Anabaena nirB gene) and nirA, which

repress the expression of the nirA operon when nitrate is absent from the culture

medium giving rise to the aforementioned nitrate effect in Anabaena sp. strain PCC

7120.

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MATERIALS AND METHODS

Strains and growth conditions. Anabaena sp. (also known as Nostoc sp.) strain

PCC 7120 was routinely grown photoautotrophically at 30°C under white light (about

25 µE s-1

m-2

), with shaking for liquid cultures. Media used for growth were BG11

(NaNO3 as the nitrogen source)(34), BG110 (BG11 without nitrate) or BG110NH4+

(BG110 supplemented with 2 mM NH4Cl and 4 mM N-tris(hydroxymethyl)methyl-2-

aminoethane sulfonic acid (TES)-NaOH buffer, pH 7.5). For growth on plates, medium

solidified with separately autoclaved 1% agar (Difco) was used. When appropriate,

antibiotics were added to plates at the following final concentrations: streptomycin

(Sm), 5 µg/ml; spectinomycin (Sp), 5 µg/ml; and neomycin (Nm), 30 µg/ml. In liquid

cultures, antibiotic concentrations used were as follows: Sm, 2 µg/ml; Sp, 2 µg/ml; and

Nm, 5 µg/ml. Strains CSE17 (19), CSE23, CSE271 and CSE272 were routinely grown

in BG110NH4+ medium supplemented with Sm and Sp. Strains EF116B and CSE27B

were routinely grown in BG110NH4+ medium supplemented with Nm. CSE27 and

CSE172 were routinely grown in BG110NH4+ medium.

For derepression experiments, cells grown in BG110NH4+ (BG110 supplemented

with 4 mM NH4Cl and 8 mM TES-NaOH buffer, pH 7.5) medium bubbled with a

mixture of air and CO2 (1% of CO2, v/v) at 30°C in the light (75 to 100 µE s-1

m-2

) were

harvested by filtration and washed with BG110 medium, resuspended in the media

indicated in each experiment and incubated under the same conditions. All media used

for derepression experiments were supplemented with 12 mM NaHCO3.

Escherichia coli DH5α, HB101, XL1-Blue and ED8654 were grown in Luria-

Bertani medium as described previously (4).

Generation of mutant strains. The method of sacB-mediated positive selection

for double recombinants in Anabaena sp. (7) was used to generate mutant strains

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EF116B (nrtB::C.K1), CSE27 (∆nirA), CSE27B (∆nirA nrtB::C.K1) and CSE23

(all0604::C.S3), and to complement mutant strain CSE17 (all0605::C.S3). Plasmids

pCSE111B (for CSE23), pCSE129 (for CSE172), pCSE142B (for CSE27), pCSE149

(for CSE271 and CSE272) and pCSE152B (for EF116B and CSE27B) were transferred

to the cyanobacterial parental strain by conjugation (11). See Table 1 for a description

of strains and plasmids. Plasmid pRL623 was used as helper plasmid in conjugations,

except for the generation of strain CSE23 for which plasmids pRL528 and pRL591-

W45 were used as helper plasmids. In all cases plasmid pRL443 was used as

conjugative plasmid. For generation of strains CSE27 and CSE172, some sucrose-

sensitive exconjugants (SmrSp

r for CSE27; Sm

rSp

rNm

r for CSE172) were grown in

BG110NH4+ liquid medium without antibiotics. These cultures were sonicated in a

cleaning bath and plated on BG110NH4+ solid medium containing 5% sucrose. Double

recombinants were identified by their sucrose-resistant antibiotic-sensitive phenotype.

In CSE27, a 666-bp internal fragment of nirA, corresponding to nucleotides 598-1263

of the 1611-nucleotide-long coding region, was deleted from the genome, and as a

consequence of the in-frame deletion of nucleotides, the modified nirA should encode a

protein of 304 amino acid residues. In all cases the genomic structure of the resultant

Anabaena mutant strain was checked by Southern analysis or PCR analysis to confirm

the absence of parental chromosomes in the resultant Anabaena strains.

DNA isolation and Southern blot analysis. Isolation of DNA from Anabaena

sp. was performed as previously described (7). For Southern blots, restriction

endonuclease-digested DNA was subjected to electrophoresis in agarose gels and

transferred to Hybond™-N+ membranes following the instructions of the manufacturer.

Labeling of probes with 32

P and hybridization was performed as described previously

(9, 21).

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RNA isolation and analysis. RNA from Anabaena sp. was prepared as

described previously (29). The resulting RNA preparations were treated with RNase-

free DNase I to eliminate contaminating DNA. For Northern blot analysis, RNA

(approximately 20-25 µg) was subjected to electrophoresis in denaturing formaldehyde

gels, transferred to Hybond™-N+ membranes, and subjected to hybridization at 65°C as

described previously (9). DNA probes (see Table 2) used in Northern experiments

were: nirA probe, PCR-generated DNA fragment using primers nir-7120-15 and nir-

7120-16 (probe a), nir-7120-23 and nir-7120-25 (probe b), or nir-7120-30 and nir-7120-

31 (probe c); narB probe, PCR-generated DNA fragment using primers N-narB-7120

and C-narB-7120; ntcA probe, NcoI/SalI DNA fragment from pCSAM61 (29); ntcB

probe, PCR-generated DNA fragment using primers Nc-ntcB and ntcB-3 (18); cnaT

probe, HincII/BstXI DNA fragment from pCSE118 (20) bearing most of the Anabaena

cnaT gene; all0603 probe, PCR-generated DNA fragment using primers all0603-1 and

all0603-2; all0604 probe, PCR-generated DNA fragment using primers all0604-1 and

all0604-2; and all0605 (nirB) probe, PCR-generated DNA fragment using primers

orf398-2 and nir-7120-10. For PCR-generated probes, Anabaena chromosomal DNA,

plasmid pCSE21 (19) or plasmid pCSE95 (18) was used as a template. Primer extension

experiments were performed as described elsewhere (4), using 20-25 µg of RNA and

primer nir-1 (19). Results were visualized and quantified with a Cyclone storage

phosphor system and OptiQuant image analysis software (Packard).

Nitrate uptake. Nitrate uptake assays were performed as previously described

(14). Ammonium-grown cells (4 to 5 µg of chlorophyll a/ml) were derepressed by

incubation for 4 h in BG11 medium. Cells were then harvested by filtration, washed

with 10 mM Tricine-NaOH buffer (pH 8.1), resuspended in the same buffer to 10 µg of

chlorophyll a/ml, and incubated under culture conditions. Uptake assays were started by

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addition of NaNO3 (100 to 110 µM, final concentration). Nitrate disappearance was

determined by estimating the concentration of nitrate in the medium in aliquots of the

cell suspensions after removal of the cells by filtration through Millipore HA 0.45-mm-

pore-size filters. Nitrate concentration was determinated by HPLC using a Partisil 10

SAX WCS Analytical Column (4.6 mm x 250 mm; 10 µm) from Whatman International

Ltd (England).

Enzyme activities. Nitrate reductase (23) and nitrite reductase (24) were

measured with dithionite-reduced methyl viologen as the reductant in cells made

permeable with mixed alkyltrimethylammonium bromide. The amount of cells added to

an enzymatic assay for nitrate reductase and nitrite reductase contained 5 and 25 µg of

chlorophyll a, respectively. Activity units correspond to µmol of nitrite produced

(nitrate reductase) or removed (nitrite reductase) per minute.

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RESULTS AND DISCUSSION

Verification of strain CSE17. Construction of mutant strain CSE17

(all0605::C.S3 or nirB::C.S3) has been previously described (19). When ammonium-

grown cells of this mutant were incubated in the absence of ammonium in cultures

bubbled with CO2-enriched air, they showed abnormally low levels of nitrite reductase

activity in nitrate-containing medium and abnormally high levels of nitrate reductase

activity in medium containing no combined nitrogen (see below). This data confirm

previously reported data (19).

To verify that the phenotype shown by strain CSE17 results from inactivation of

all0605 (nirB) and not from any additional mutation elsewhere in the genome, the

mutated version of nirB in CSE17 was replaced by a wild-type version of the gene (see

Materials and Methods for details). In strain CSE172, the genomic structure in the nirB

region was identical to that of the wild-type strain PCC 7120 (not shown). Nitrate and

nitrite reductase levels in strain CSE172 were similar to those of the wild type rather

than to those of its parental strain CSE17 showing that the phenotype of strain CSE17

results from the inactivation of nirB.

Two small ORFs, all0604 and all0603, whose coding sequences overlap by 3

nucleotides, are located downstream of all0605 (Fig. 1). The gene-cassette inserted into

all0605 in strain CSE17, C.S3 (12), contains transcriptional terminators that are

effective in Anabaena sp. strain PCC 7120 (see, e. g., 19). To verify that the phenotype

of strain CSE17 results from inactivation of all0605 and not from a polar effect on a

downstream gene, the expression of all0604-all0603 was investigated and a mutant of

all0604 was constructed with the same cassette, C.S3 (see Fig. 1 and Materials and

Methods for details). Mutant strain CSE23 (all0604::C.S3) was able to grow in media

containing ammonium, nitrate or no combined nitrogen and exhibited nitrate reductase

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and nitrite reductase activity levels that were similar (within 10 %) to those of the wild-

type strain. Additionally, the expression of nirA and narB in mutant strain CSE23,

investigated by Northern analysis, showed no appreciable difference to that observed in

the wild-type strain PCC 7120 (not shown).

To investigate the expression of all0604 and all0603, Northern analysis was

performed with probes of each of these ORFs and RNA isolated from strains PCC 7120,

CSE17 and CSE172. The all0604 probe hybridized to bands of about 0.33 kb (likely

corresponding to an all0604 transcript) and 0.87 kb (likely corresponding to a transcript

covering both all0604 and all0603) (Fig. 2). Only the latter band was obtained using an

all0603 probe and no expression of all0603 was observed in mutant strain CSE23

(all0604::C.S3) (not shown). Thus, a bicistronic transcript is produced, but a

monocistronic transcript is also observed that may result from premature transcription

termination downstream of all0604. The 0.87-kb bicistronic transcript accumulated at

an increased level after 24 h incubation in the absence of combined nitrogen. No

significant differences in the expression profiles of all0603 and all0604 were observed

between strains PCC 7120, CSE17, and CSE172 (Fig. 2). The results of analysis of the

nirB mutant (all0605::C.S3) and of expression of all0604-all0603 both indicate that the

phenotype of CSE17 cannot be ascribed to a polar effect on the expression of

downstream genes.

Effect of inactivation of nirB on the expression of nitrate assimilation

enzyme activities and genes. The development of the nitrate and nitrite reductase

activities was analyzed in cultures of wild-type strain PCC 7120 and mutant strain

CSE17 during a 24-h induction experiment in nitrate-containing media. In the wild type

(Fig. 3A), both enzymatic activities reached a plateau after 6 h of incubation in nitrate-

containing media, with activities of about 89 mU (mg of protein)-1

for nitrate reductase

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and about 23 mU (mg of protein)-1

for nitrite reductase. In the mutant (Fig. 3B), the

nitrite reductase activity also reached a plateau at 6 h, showing an activity of about 11

mU (mg of protein)-1

, whereas the nitrate reductase activity increased during the course

of the experiment to reach an activity of about 131 mU (mg of protein)-1

at 24 h. An

accumulation of nitrite in the culture medium was observed specifically for strain

CSE17 indicating that its nitrite reductase is unable to reduce all the nitrite produced by

nitrate reductase, the excess nitrite being excreted.

The development of nitrate reductase in strains PCC 7120, CSE17 and CSE172

was also studied in media lacking combined nitrogen (Fig. 4). Expression was observed,

and after reaching a peak shortly after the beginning of the incubation, the activity

decayed in the wild type but much less in strain CSE17. Strain CSE172 behaved like the

wild type. The level of nitrate reductase activity after 24 h in the absence of nitrate was

about 15-fold higher in mutant CSE17 than in the wild type. These results show that

inactivation of nirB has profound effects on the enzyme activities in the nitrate

assimilation system resulting in low levels of nitrite reductase and, especially when the

cells are incubated in the absence of nitrate, abnormally high levels of nitrate reductase.

To test whether the abnormal levels of nitrate and nitrite reductase activities

shown by strain CSE17 resulted from an altered expression of the nirA operon,

Northern analyses were carried out with probes of the nirA and narB genes.

Hybridizations were performed with total RNA isolated from cells of strains PCC 7120,

CSE17, and CSE172 incubated under different nitrogen regimes. As previously reported

for the nirA operon (19), which produces a long transcript of close to 10 kb that is

unstable, only a smear of degraded RNA products could be detected with both the nirA

and narB probes (Fig. 5). In strains PCC 7120 and CSE172, a high level of expression

of narB took place only in media without ammonium in the presence of nitrate. In the

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nirB mutant (strain CSE17), however, a high level of expression was observed in the

absence of ammonium both in the presence and absence of nitrate (Fig. 5A). A similar

expression profile was obtained with the nirA probe (Fig. 5C). Levels of the 5’ region of

the nirA operon transcript in strain CSE17 were also analyzed by primer extension. The

obtained data (Fig. 6) corroborated those obtained by Northern experiments, suggesting

that the increased nirA operon transcript levels observed in strain CSE17 in the absence

of combined nitrogen corresponded to a higher utilization of the nirA operon promoter

in this strain.

To test whether the expression of the positive regulatory elements NtcA, NtcB

and CnaT was affected in mutant strain CSE17, Northern analysis was performed. The

expression patterns observed for the ntcA, ntcB and cnaT genes under the different

nitrogen regimes tested were similar in strains PCC 7120, CSE17 and CSE172 (Fig. 5)

and similar to those previously reported (18, 20, 29). These results indicate that NirB is

not involved in the regulation of transcription of these genes and, additionally, that the

altered levels of nirA operon expression observed in strain CSE17 do not result from an

altered expression of the positive regulatory elements NtcA, NtcB and CnaT. Therefore,

NirB appears to be a factor inhibiting the expression of the nirA operon, especially in

the absence of nitrate, in Anabaena sp. strain PCC 7120.

The accumulation of nirA operon transcripts in strain CSE17, observed with

probes of nirA and narB, explains the higher nitrate reductase activity observed in this

mutant in both the presence and absence of nitrate; however, it does not explain the low

levels of nitrite reductase activity observed in the presence of nitrate. A similar

phenotype of expression of low levels of nitrite reductase activity has been observed for

a nirB mutant of S. elongatus, although no effect of the corresponding mutation on the

nirA operon transcript level has been reported (35). It has been proposed that NirB is

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required as a chaperone or scaffold for expression of maximum nitrite reductase activity

(35). Similarity of NirB to CpcE and NblB is based on the presence in these proteins of

a number of HEAT-like repeats, which consist of tandemly repeated amino acid

modules that appear to function in protein-protein interactions and may have a

scaffolding role (22). Anabaena NirB appears to have six such HEAT repeats. A role

based on the presence of these structural motifs would explain why NirB from S.

elongatus and Anabaena sp. strain PCC 7120, two homologous proteins that appear to

have a similar function in nitrate reduction and are therefore orthologues, show such a

low identity degree (23 %). If NirB works through protein-protein interactions, it is

conceivable that NirB interacts with the nitrite reductase allowing it to reach a

maximally active conformation for the development of its enzymatic activity. In turn,

this raises the possibility of a regulation of the nirA operon by nitrite reductase itself

rather than by NirB.

Study of a nirA mutant. Different mutant strains in the Anabaena nirA operon

generated by lux-tagged Tn5 transposon mutagenesis have been described (6). One of

those mutants, strain TLN10 (nirA::Tn5), shows a phenotype similar to that of the nirB

mutant. When cells are transferred from ammonium-containing medium to medium

lacking combined nitrogen, a luciferase activity develops in strain TLN10 that is very

high as compared to that showed by the mutant strains TLN12 (nrtC::Tn5) and TLN21

(Tn5 inserted in the nrtD-narB intergenic region) (6). Given that in both cases, the nirB

mutant and strain TLN10, the nitrite reductase activity is affected (we assume that the

Tn5 insertion inactivates nitrite reductase), we decided to mutate the nirA gene in order

to check whether the nitrite reductase is involved in the regulation of expression of the

nirA operon.

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A nirA mutant, strain CSE27, was constructed by deleting in frame a 666-bp

internal fragment of nirA, corresponding to nucleotides 598-1263 of the 1611-

nucleotide-long coding region (see Fig. 1 and Materials and Methods for details). When

nitrate was used as a nitrogen source, strain CSE27 was unable to grow and nitrite

accumulated in the culture medium (not shown). The expression of nirA in mutant strain

CSE27 was investigated by Northern analysis using RNA isolated from strains PCC

7120, CSE17 and CSE27 (Fig. 7). As is the case for strain CSE17, a high expression of

the nirA operon in the absence of ammonium in both the presence and absence of nitrate

was observed in strain CSE27, and the observed levels of expression were even higher

than in strain CSE17.

To verify that the phenotype shown by strain CSE27 resulted from inactivation

of nirA, a wild-type version of the nirA gene bearing the complete nirA operon promoter

was introduced into strain CSE27. Plasmid pCSE149 (see Table 1), a derivative of

pCSEL24 (31), can recombine with the nirA region in the chromosome and with the

nucA region in the Anabaena alpha megaplasmid. In the exconjugant strains CSE271

and CSE272, pCSE149 was integrated into the alpha megaplasmid (Fig. 8A) and into

the chromosome (Fig. 8B), respectively, in such a way that in both cases one copy of

the nirA operon was maintained as in CSE27 and an intact nirA gene together with its

promoter was additionally present. The genomic structure of these strains was

confirmed by PCR analysis using different primer pairs (nui-7120-4 and nir-7120-28;

nir-7120-27 and nrtA-7120-2; nir-7120-6 and orf398-4; ntcs3 (19) and nrtA-7120-2;

ntcs3 and nrtA-7120-3; see Table 2). Both strains were able to grow using nitrate as a

nitrogen source. The expression of narB was investigated by Northern analysis using

total RNA isolated from strains CSE271 and CSE272 and, as controls, PCC 7120 and

CSE27 (Fig. 8C). In contrast to strain CSE27, strains CSE271 and CSE272 presented an

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expression of narB similar to that shown by strain PCC 7120. These results indicate that

the introduction of a wild-type copy of nirA into strain CSE27 restored a wild-type

phenotype of nirA operon expression at the mRNA level. Using the DNA fragment

deleted in CSE27 as a nirA probe, we were also able to corroborate an ammonium- and

nitrate-regulated expression of the wild-type copy of nirA introduced in strains CSE271

and CSE272 (Fig. 8D). Interestingly, however, the nirA copy in the alpha megaplasmid

(strain CSE271) was expressed at higher levels than that present in the chromosome

(strain CSE272).

As described in the Introduction, a high level of expression of the nirA operon in

Anabaena sp. strain PCC 7120 requires both the absence of ammonium and the

presence of nitrate or nitrite, which act as inducers, in the culture medium. The results

described above suggest that nitrite reductase has, in addition to its role in nitrite

reduction to ammonium during nitrate assimilation, a negative role in the expression of

the nirA operon when this cyanobacterium is incubated in the absence of both

ammonium and nitrate in the culture medium. A simple interpretation of our results

would be that in the nirB and nirA mutants, which have low levels or lack nitrite

reductase, respectively, nitrite accumulates in the cells up to levels that strongly induce

the expression of the nirA operon. However, whereas this interpretation would be

evident for those cultures supplemented with nitrate, we observed similarly increased

nirA operon mRNA levels in the nirB and nirA mutants incubated in the absence as well

as in the presence of added nitrate. The possibility remains, nevertheless, that traces of

nitrate or nitrite present in the BG110 medium are concentrated within the cells to act as

inducers.

Mutants of the NrtABCD transporter. An Anabaena ABC-type nitrate

transporter, NrtABCD, has been previously identified (19). To check whether nitrate

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concentrated within the cells from traces present in the medium could be acting as an

inducer, the nrtB gene, which encodes the transmembrane component of the nitrate

transporter, was mutated in strain CSE27 and in its parental strain EF116 (see Table 1

for details). For this, the C.K1 cassette, which confers resistance to neomycin and does

not bear transcriptional terminators, was introduced into the ScaI restriction site of nrtB

in the same orientation as this gene (see Fig. 1 and Materials and Methods for details).

Two mutants, strains EF116B (nrtB::C.K1) and CSE27B (∆nirA, nrtB::C.K1), were

obtained that were defective in the active transport of nitrate (Fig. 9A). The expression

of the nirA gene was studied by Northern analysis, and both mutants presented an

expression profile similar to that shown by their respective parental strains (Fig. 9B).

The fact that similar expression levels were observed in the presence and absence of

added nitrate in spite of the absence of an active transporter makes it unlikely the

possibility of a role of traces of nitrate in regulation.

The Anabaena NrtABCD transporter also mediates the uptake of nitrite into the

cells (ref. 32 and our unpublished results), but nitrite can enter into the cyanobacterial

cells also by means of diffusion of nitrous acid (16). Diffusion is not however a

mechanism that could concentrate nitrite within the cells, and direct determination of

nitrite in cell extracts from filaments incubated without added nitrate under the

conditions described in this work rendered negligible levels of nitrite (not shown). Our

results argue against accumulation of an inducer (nitrate or nitrite) as responsible for the

increased levels of the expression of the nirA operon observed in nirB and nirA mutants.

They suggest instead that NirA (the nitrite reductase) and NirB (a possible nitrite

reductase chaperone) somehow inhibit the expression of the nirA operon when the wild-

type Anabaena filaments are incubated in the absence of both ammonium and nitrate.

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Concluding remarks. As mentioned above, both Anabaena mutants, nirA and

nirB, have lost the nitrate effect on expression of the nirA operon (see Fig. 7). We do

not know whether the observed phenotype of the nirB mutant just results from the lack

of NirB protein or is related to the possible role of NirB as scaffolding protein in the

maturation of the nitrite reductase. If the latter were the case, the nitrite reductase would

be the key element in the negative regulation of the nirA operon in the absence of

ammonium. How nitrite reductase could exert this negative role remains to be

elucidated. A plausible hypothesis is that the nitrite reductase acts as a modulator of a

transcription factor. Because NtcA is a general N-control transcription factor and NtcB

is a nitrate assimilation pathway-specific transcriptional activator, NtcB could be

favored as a candidate target for nitrite reductase. Indeed, a role of NtcB mediating a

nitrite effect in the expression of the nirA operon in S. elongatus has been suggested (1).

The nitrite reductase could switch from being an inhibitor of the NtcB transcriptional

activator in the absence of nitrate or nitrite to acting as an enzyme in the presence of

these nitrogen sources. It has recently been found that some enzymes, termed ‘trigger

enzymes’, can control gene expression in response to the availability of their substrates

through different mechanisms: by binding to either DNA or RNA or by modulating of

the activity of transcription factors through either covalent modification or protein-

protein interactions (8). Our results suggest that nitrite reductase from Anabaena sp.

strain PCC 7120 is a ‘trigger enzyme’ that, in addition to its role in nitrite reduction to

ammonium, exerts a regulatory role on the nitrate assimilation system.

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ACKNOWLEDGMENTS

We thank A. M. Muro-Pastor and A. Valladares for their help during this work

and A. Herrero for discussion. Use of DNA sequences from the DOE-Joint Genome

Institute (USA) and Kazusa DNA Research Institute (Japan) databases is acknowledged.

This work was supported by grant numbers BFU2005-07672 and BFU2008-03811 from

the Ministerio de Ciencia y Tecnología (Spain).

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Figure legends

FIG. 1. Genomic region of Anabaena sp. strain PCC 7120 bearing the nitrate

assimilation gene cluster. Genes and ORFs are indicated by thick arrows, which also

show the direction of transcription. Black arrows correspond to the ORFs investigated

in this work. The location of the restriction sites into which gene-cassettes (C.S3 for

strains CSE17 and CSE23; and C.K1 for strains EF116B and CSE27B) were inserted is

indicated. The region deleted from nirA in strain CSE27 is indicated as a ruled bar.

Abbreviations for some restriction endonuclease sites: B, BglI; C, ClaI; E5, EcoRV; H,

HindIII; P, PvuII; S, SpeI; Sc, ScaI; X, XbaI. Horizontal lines below the genes denote

probes used for Northern analyses.

FIG. 2. Northern analysis of the expression of all0604 in strains PCC 7120,

CSE17, and CSE172. Hybridization assays were carried out using RNA isolated from

cells grown with ammonium (NH4+) or grown with ammonium and incubated for 4 or

24 h in medium containing nitrate (NO3-) or no combined nitrogen (-N). Hybridization

to rnpB (40) served as a loading and transfer control (lower panel). The position and

size (indicated in kb) of the detected transcripts is shown on the left.

FIG. 3. Changes in levels of nitrate and nitrite reductase activities, and nitrite

accumulation in the culture medium after transfer of ammonium-grown cells to nitrate-

containing medium. (A) Strain PCC 7120 (WT); (B) strain CSE17. Nitrate reductase

activity (diamonds); nitrite reductase activity (squares); nitrite concentration in the

medium (triangles).

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FIG. 4. Changes in levels of nitrate reductase activity after transfer of

ammonium-grown cells to a medium containing no combined nitrogen. Strain PCC

7120 (circles); strain CSE17 (squares); strain CSE172 (triangles).

FIG. 5. Northern analysis of the expression of the nirA operon and of some

regulatory genes in strains PCC 7120, CSE17, and CSE172. Hybridization assays were

carried out using RNA isolated from cells grown with ammonium (NH4+) or grown with

ammonium and incubated for 4 or 24 h in medium containing nitrate (NO3-) or no

combined nitrogen (-N). The hybridization probes used (see Materials and Methods for

details) corresponded to narB (A), cnaT (B), nirA (C; fragment “a” in Fig. 1), ntcA (D)

or ntcB (E). Hybridization to rnpB (40) served as a loading and transfer control for each

of the two filters used. The position of some size markers (A, C) or identified transcripts

(B, D, E) is shown, indicated in kb, on the left.

FIG. 6. Primer extension analysis of the expression of the nirA gene in strains

PCC 7120 and CSE17. Primer extension assays were carried out using oligonucleotide

nir-1 as a primer and RNA isolated from cells grown with ammonium (NH4+) or grown

with ammonium and incubated for 4 h in medium containing nitrate (NO3-) or no

combined nitrogen (-N). The arrowhead points to the extension product identifying the

Anabaena nirA operon tsp. The sequencing ladders presented were generated with the

same primer used in the primer extension reactions and plasmid pCSE26 as template.

FIG. 7. Northern analysis of the expression of nirA in strains PCC 7120, CSE17,

and CSE27. Hybridization assays were carried out using RNA isolated from cells grown

with ammonium (NH4+) or grown with ammonium and incubated for 4 in medium

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containing nitrate (NO3-) or no combined nitrogen (-N). Fragment “b” in Fig. 1 was

used as nirA probe (see Materials and Methods for details). Hybridization to rnpB (40)

served as a loading and transfer control (lower panel). The position and size (indicated

in kb) of some size standards is shown on the left. The small changes in the pattern of

hybridization bands in strain CSE27 as compared to strains CSE17 and PCC 7120 result

from the deletion of 666 bp from nirA in CSE27.

FIG. 8. Genomic structure of nirA complementing constructs in strains CSE271

and CSE272 and Northern analysis of the expression of narB and nirA in strains PCC

7120, CSE27, CSE271 and CSE272. The complementing constructs included ORF

all0606, the nirA operon promoter region and the nirA gene incorporated into the nucA-

nuiA region of the alpha megaplasmid (A) or in the chromosomal nirA region (B).

Hybridization assays were carried out using RNA isolated from cells of the indicated

strains grown with ammonium (NH4+) or grown with ammonium and incubated for 4 in

medium containing nitrate (NO3-) or no combined nitrogen (-N). The hybridization

probes used (see Materials and Methods) corresponded to narB (C) and nirA (D;

fragment “c” in Fig. 1). Hybridization to rnpB (40) served as a loading and transfer

control. The position and size (indicated in kb) of some size standards is shown on the

left.

FIG. 9. Nitrate uptake and Northern analysis of the expression of nirA in strains

PCC 7120, EF116B, CSE27 and CSE27B. (A) Ammonium-grown cells of strains

PCC7120 (closed diamonds), EF116B (open diamonds), CSE27 (closed triangles) and

CSE27B (open triangles) were washed, resuspended in BG11 medium (17,6 mM

NaNO3), and incubated for 4 h as indicated in Materials and Methods for derepression

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of the nitrate assimilation system. Cells were then harvested and used for nitrate uptake

assays (see Materials and Methods). At the indicated times, nitrate was measured by

HPLC using aliquots withdrawn from the assay mixture. (B) Hybridization assays were

carried out using RNA isolated from cells of the indicated strains grown with

ammonium (NH4+) or grown with ammonium and incubated for 4 in medium containing

nitrate (NO3-) or no combined nitrogen (-N). Fragment “b” in Fig. 1 was used as nirA

probe (see Materials and Methods). Hybridization to rnpB (40) served as a loading and

transfer control (lower panel). The position and size (indicated in kb) of some size

standards is shown on the left.

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Table 1. Cyanobacterial strains and plasmids used in this study.

Strain or

plasmid

Relevant characteristics Reference

Strains

PCC 7120 Wild-type Anabaena strain (34)

EF116 Derivative of Anabaena sp. strain PCC 7120 unable to fix nitrogen under aerobic conditions (41)

EF116B Nmr derivative of strain EF116; nrtB::C.K1 This work

CSE17 SmrSp

r derivative of strain PCC 7120; all0605::C.S3 (19)

CSE172 SmsSp

s derivative of strain CSE17; all0605::C.S3 substituted by a wild-type version of

all0605

This work

CSE23 SmrSp

r derivative of strain PCC 7120; all0604::C.S3 This work

CSE27 Derivative of strain EF116; ∆nirA This work

CSE27B Nmr derivative of strain CSE27; ∆nirA nrtB::C.K1 This work

CSE271 SmrSp

r derivative of strain CSE27; pCSE149 plasmid integrated into nucA region of the

alpha megaplasmid

This work

CSE272 SmrSp

r derivative of strain CSE27; pCSE149 plasmid integrated into nirA region of the

Anabaena chromosome

This work

Plasmids

pCSE111B 2.79-kb SpeI fragment from pCSE73 (19), bearing AccI-ended gene-cassette C.S3 (12) inserted into the site ClaI of all0604, cloned in pRL278; used to generate mutant strain

CSE23

This work

pCSE129 1.94-kb XbaI/HindIII fragment from pCSE26 (19), that contains part of all0605 and the

whole all0606, cloned in pRL278; used to complement mutant strain CSE17

This work

pCSE142 1166-bp product of PCR with primers nir-7120-23, nir-7120-25, nir-7120-26 and nrtA-7120-

3, with pCSE26 as template; presents a deletion of a 666-bp internal segment of nirA,

corresponding to nucleotides 598-1263 of the 1611-nucleotide-long coding region

This work

pCSE142B 1.3-kb PvuII/SalI fragment from pCSE142 between the sites NruI/XhoI of pRL277; used to

generate mutant strain CSE27

This work

pCSE152B 2.9-kb ClaI/XbaI fragment from pCSE2 (19), bearing HincII-ended gene-cassette C.K1 (12)

inserted into the ScaI site of nrtB (in the same direction as nrtB), cloned in pRL277; used to

generate mutant strains EF116B, CSE17B and CSE27B

This work

pCSE149 5.3-kb EcoRV/PvuII fragment from pCSE78B (19) inserted between the sites EcoRV/NruI

of pCSEL24(31); used to complement mutant strain CSE27

This work

pRL277 SmrSp

r, sacB-carrying, mobilizable vector (5)

pRL278 Nmr, sacB-carrying, mobilizable vector (5)

pRL443 Kms derivative of conjugative plasmid RP4 (11)

pRL528 Mobilization-helper; encodes M.AvaI and M.Eco47II. (11)

pRL591-W45 Mobilization-helper; encodes M.EcoT22I (10)

pRL623 Mobilization-helper; encodes M.AvaI, M.Eco47II, and M.EcoT22I (10)

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Table 2.

Oligodeoxynucleotide primers used in this work

Primer Sequence (5’-3’) nir-7120-6 GGT GTT GGT CGT GGG TAC

nir-7120-10 GCA AGC GAT CGC ACT GCC

nir-7120-15 GCA ACA GAC CGA GAT CAT CG

nir-7120-16 CCC CAT TCA TCA ATT AGC C

nir-7120-23 CTA CCC CCA AAG CCA GCC TC

nir-7120-25 GAG GGC GAA CGC ATG AAC TGA ATT ATC CC

nir-7120-26 ACG TTC ATG CGT TCG CCC TCA TCG AAA CC

nir-7120-27 CGG GAT AAT TCA GTT CAT GC

nir-7120-28 TTC CGG CAC GGG CGC ACA ATT TGG CAA CTT CG

nir-7120-30 GGA AAT CAA CGA TTT AGC CTT TGT TCC

nir-7120-31 GTT GCA AAA TTG TGC GCC CGT G

orf398-2 CAT AGT GCA GAT GAT TTG TC

orf398-4 TCA GTG CAG CGA TCG CAT GG

nrtA-7120-2 TCC AAT CTT GCC GCA TAC

nrtA-7120-3 TCT AGA GGA AGT ACA GCC ATG TAC C

N-narB-7120 GGA GCG AAG CGA CGT GAC

C-narB-7120 GGT CAG TTG GGT AAA CTC

all0603-1 CGA AGC CAT TTG ATG AAC

all0603-2 CAA TCG AAC TGA GAA ATC AC

all0604-1 AAT GAC CTG GGA GGT AGA G

all0604-2 TCA TAG GAA CCC CTC TG

nui-7120-4 ATG AGT GAG TCT GAA TAC CC

Introduced restriction enzyme cutting sites are shown in bold face.

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B

Fig.1

(Frías and Flores)

E5

(CSE23) C.S3

HC PS XS

cnaT ntcB all0603 all0605 all0606 nirA nrtA nrtB nrtC nrtD narB

all0604 (nirB)

C.S3 (CSE17)

1 kbCSE27Sc

C.K1 (EF116B/CSE27B)

(a)(b)

(c)

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24 h NO3-

NH4+

4 h NO3-

4 h -N

24 h -N

PC

C 7

12

0 C

SE

17

CS

E17

2

24 h NO3-

NH4+

4 h NO3-

4 h -N

24 h -N

24 h NO3-

NH4+

4 h NO3-

4 h -N

24 h -N

0.8

7

0.3

3

Fig

. 2

(Frías an

dF

lores)

(all0

604

)

(rnp

B)

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0

20

40

60

80

100

120

140

0 5 10 15 20 25

time (h)

0

100

200

300

400

500

600

700

nit

rite

( µµ µµM

)

En

zym

ati

cact

ivit

y

(mU

/mg

pro

t)

20

40

60

80

100

120

140

100

200

300

400

500

600

700

nit

rite

( µµ µµM

)

A

B

En

zym

ati

cact

ivit

y

(mU

/mg

pro

t)

Fig. 3

(Frías and Flores)

WT

CSE17

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0

10

20

30

40

50

60

0 5 10 15 20 25time (h)

En

zym

ati

cact

ivit

y

(mU

/mg

pro

t)

Fig. 4

(Frías and Flores)

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Fig. 5

(Frías and Flores)

PCC 7120 CSE17 CSE172

24 h

NO

3-

NH

4+

4 h

NO

3-

4 h

-N

24 h

-N

24 h

NO

3-

NH

4+

4 h

NO

3-

4 h

-N

24 h

-N

24 h

NO

3-

NH

4+

4 h

NO

3-

4 h

-N

24 h

-N

A

2.82.3

1.5

0.5

(narB)

C

2.82.31.5

0.5

(nirA)

0.95

E(ntcB)

D

1.5

(ntcA)

B

1.1

(cnaT)

(rnpB)

(rnpB)

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NH

4+

NO

3-

-N

PCC 7120 CSE17

NH

4+

NO

3-

-NA C G T

Fig. 6

(Frías and Flores)

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Fig. 7

(Frías and Flores)

NO

3-

-N NH

4+

PCC 7120 CSE17 CSE27

2.8

2.3

1.5

NO

3-

-N NH

4+

NO

3-

-N NH

4+

(nirA)

(rnpB)

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Fig. 8

(Frías and Flores)

1 kb

BE5

C.S3

nuiA nucA nirA all0606 nuiA nucA

X

(Alpha megaplasmid)

A6

7

B E5P X B

C.S3

E5XCSE27

all0605 all0606 nirA nucA nuiA all0606 nirA nrtA nrtB nrtC nrtD narB

B (Chromosome)

3

54

1

2

8

PCC 7120 CSE27 CSE271 CSE272

NO

3-

-N NH

4+

NO

3-

-N NH

4+

NO

3-

-N NH

4+

NO

3-

-N NH

4+

2.82.3

1.5

C(narB)

2.82.3

1.5

PCC 7120 CSE27 CSE271 CSE272

NO

3-

-N NH

4+

NO

3-

-N NH

4+

NO

3-

-N NH

4+

NO

3-

-N NH

4+

D(nirA)

(rnpB) (rnpB)

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Fig. 9

(Frías and Flores)

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70

Time (min)

Nit

ra

te (µµ µµ

M)

A

PCC 7120 EF116B CSE27 CSE27B

NO

3-

-N NH

4+

NO

3-

-N NH

4+

NO

3-

-N NH

4+

NO

3-

-N NH

4+

2.8

2.3

1.5

B

(nirA)

(rnpB)

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